HK1119061A

HK1119061A - Nucleic acid constructs

Info

Publication number: HK1119061A
Application number: HK08110702.0A
Authority: HK
Inventors: James Fuller
Original assignee: Powderject Vaccines, Inc.
Priority date: 2005-02-01
Filing date: 2006-02-01
Publication date: 2009-02-27

Description

Nucleic acid constructs

Technical Field

The present invention relates to the fields of molecular biology and immunology, and in particular to reagents for use in nucleic acid immunization techniques. More particularly, the invention relates to nucleic acid constructs expressing polypeptides, particularly antigenic polypeptides, particularly influenza antigens, as well as nucleic acid constructs expressing adjuvant polypeptides, and to methods of nucleic acid immunization using such agents.

Background

Gene therapy and nucleic acid vaccination are promising approaches for the treatment and prevention of acquired and inherited diseases. Such techniques enable the transfer of a desired nucleic acid into a subject and subsequent expression in vivo. Transfer can be accomplished by transfecting cells or tissues from the subject in vitro and reintroducing the transformed material into the host. Alternatively, the nucleic acid may be administered directly into the subject.

Each of these techniques requires the efficient expression of the nucleic acid in transfected cells to provide a sufficient amount of the therapeutic or antigenic gene product. Several factors are known to influence the level of expression obtained, including transfection efficiency, and the efficiency of transcription of the gene or sequence of interest and translation of mRNA.

A variety of expression systems have been described in the art, each generally consisting of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. Such control sequences include transcriptional promoter sequences and transcriptional initiation and termination sequences. Promoters commonly used in mammalian cell expression systems include the SV40 early promoter, the Cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter (Chapman et al (1991) Nucl. acids Res.19: 393979-. Non-viral promoters, such as those derived from the murine metallothionein gene, are also commonly used.

Expression systems typically include transcriptional regulatory elements, referred to as "enhancers". Enhancers are broadly defined as cis-acting sequences (cis-acting agents) that, when operably linked to a promoter/gene sequence, will increase the transcription of the gene sequence. Enhancers function effectively closer to the sequence of interest than other expression control elements Much more (e.g.promoters) and can be located in any orientation of the sequence under investigation (Banerji et al (1981) Cell27：299-308，deVilleirs et al.(1981)Nucl.Acids Res.9: 6251-6264). Enhancers have been identified from a variety of viral sources, including polyoma virus (polyoma virus), BK virus, Cytomegalovirus (CMV), adenovirus, Simian Virus 40 (SV 40), Moloney sarcoma virus (Moloney sarcoma virus), bovine papilloma virus (bovine papilloma virus) and Rous sarcoma virus (Rous sarcoma virus) (devillirs et al supra, Rosenthal et al (1983) Science222：749-755，Hearing et al.(1983)Cell 33：695-703，Weeks et al.(1983)Mol.Cell.Biol.3：1222-1234，Levinson et al.(1982)Nature295: 568-572, and Luciw et al (1983) Cell33：705-716)。

Many expression systems for nucleic acid immunization and gene therapy use the hCMV immediate early promoter. See, e.g., U.S. patent nos. 5168062 and 5385839, and european patent specification 0323997B1, to Stinski. Expression vectors using the hCMV immediate early promoter include, for example, pWRG7128(Roy et al, Vaccine, 764-778, 2001) and pBC12/CMV and pJW4303 (mentioned in WO 95/20660). The Chapman et al (1991) described above have reported: in the absence of hCMV intron a, the level of hCMV immediate early promoter expression decreased.

Disclosure of Invention

Nucleic acid constructs have been developed that provide increased expression of heterologous coding sequences in host cells using operable viral promoter/expression sequences. The construct is suitable for the efficient expression of genes, particularly antigen-encoding genes, and thus can be used for nucleic acid immunization. The constructs may also be used to express adjuvant polypeptides. The construct may be provided to a vector particle for particle-mediated nucleic acid immunization. In a particularly preferred embodiment of the invention, the construct encodes an immunogenic variant of an influenza antigen, an immunogenic fragment thereof, or both. In a further particularly preferred embodiment, the construct encodes an adjuvant polypeptide.

Accordingly, the present invention provides a nucleic acid construct suitable for delivery to a subject to induce an immune response against an influenza virus Hemagglutinin (HA) antigen, the construct comprising:

(i) a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

(b) exon 1 and at least a portion of exon 2 of the hCMV major immediate early gene; and

(c) a heterologous intron to replace the intron a region of the hCMV major immediate early gene;

(ii) a coding sequence operably linked to a chimeric promoter, wherein said coding sequence encodes an influenza virus Hemagglutinin (HA) antigen, an immunogenic fragment thereof, or an immunogenic variant of said antigen or fragment, said variant having at least 80% amino acid homology to said antigen or fragment;

(iii) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence and operably linked to the chimeric promoter; and

(iv) an enhancer sequence derived from the 3 'untranslated region (UTR) of the HBsAg sequence or the 3' UTR of the simian CMV immediate early gene sequence, operably linked to the chimeric promoter, and located downstream of the coding sequence.

The present invention also provides a nucleic acid construct suitable for delivery to a subject to induce an immune response against an influenza virus Hemagglutinin (HA) antigen, the construct comprising:

(i) a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

(c) a heterologous intron to replace the intron a region of the hCMV major immediate early gene; and

(ii) a coding sequence operably linked to said chimeric promoter, wherein said coding sequence encodes an influenza virus Hemagglutinin (HA) antigen, an immunogenic fragment thereof, or an immunogenic variant of said antigen or fragment, said variant having at least 80% amino acid homology to said antigen or fragment.

In addition, the present invention provides a nucleic acid construct comprising a chimeric promoter sequence and a cloning site for insertion of a coding sequence operably linked to the chimeric promoter, wherein the chimeric promoter sequence comprises:

(a) hCMV immediate early promoter sequence;

(c) A heterologous intron to replace the intron a region of the hCMV major immediate early gene.

In a preferred embodiment, the construct comprises a coding sequence inserted into a cloning site. Thus, the coding sequence may be provided at the cloning site.

In a further preferred embodiment, the construct of the invention may further comprise:

(a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV type 2gD antigen sequence and operatively linked to the chimeric promoter; and/or

(b) An enhancer sequence derived from the 3 'untranslated region (UTR) of the HBsAg sequence, or the 3' UTR of the simian CMV immediate early gene sequence, and operably linked to the chimeric promoter, and the enhancer sequence is located downstream of the cloning site.

The invention also provides a nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to the promoter, wherein the construct further comprises:

(a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence, operably linked to a coding sequence and a promoter, said promoter being heterologous to said coding sequence; and/or

(b) An enhancer sequence located 3 'of the coding sequence and operably linked thereto, wherein the enhancer sequence is derived from the 3' UTR of the HBsAg sequence or the 3 'UTR of the simian CMV immediate early gene sequence and the coding sequence is heterologous to the 3' enhancer sequence.

The present invention also provides a nucleic acid construct comprising:

(i) a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

(ii) a cloning site for inserting a coding sequence operably linked to the chimeric promoter; and

(iii) (a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence, operably linked to a chimeric promoter; and/or

(b) An enhancer sequence derived from the 3 'untranslated region (UTR) of the HBsAg sequence, or the 3' UTR of the simian CMV immediate early gene sequence, and operably linked to the chimeric promoter, wherein the enhancer sequence is located downstream of the cloning site.

The present invention also provides a nucleic acid construct comprising:

(i) a promoter sequence;

(ii) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence; and

(iii) a coding sequence operably linked to (i) and (ii)

Wherein the coding sequence is heterologous to the untranslated leader sequence.

The present invention also provides a nucleic acid construct comprising:

(i) a promoter sequence;

(ii) (ii) a coding sequence operably linked to the promoter sequence (i); and

(iii) (iii) an enhancer sequence located 3' to the coding sequence (ii) and operably linked thereto;

wherein the enhancer sequence (iii) is derived from the 3 ' UTR of an HBsAg sequence or the 3 ' UTR of a simian CMV immediate early gene sequence and the coding sequence (ii) is heterologous to the 3 ' enhancer sequence.

(a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence, operably linked to said coding sequence and a promoter, said promoter being heterologous to said coding sequence; and/or

(b) An enhancer sequence located 3 'of the coding sequence and operably linked thereto, wherein the enhancer sequence is derived from the 3' UTR of an HBsAg sequence or the 3 'UTR of a simian CMV immediate early gene sequence and the coding sequence is heterologous to the 3' enhancer sequence.

The invention further provides a population of nucleic acid constructs, wherein the population comprises at least two different constructs of the invention.

In another example, the invention provides a purified and isolated chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

The invention also provides

-a method of expressing a polypeptide of interest in a mammalian cell, which method comprises transferring a nucleic acid construct, population of nucleic acid constructs or coated particles of the invention into said cell;

-coated particles suitable for delivery by a particle-mediated delivery device, the coated particles comprising vector particles coated with a nucleic acid construct of the invention comprising a coding sequence encoding said polypeptide;

-a dose container for a particle-mediated delivery device comprising said coated particles;

-a particle-mediated delivery device loaded with said coated particles;

a method of nucleic acid immunization comprising administering to a subject an effective amount of the coated particles of the invention;

-a pharmaceutical composition comprising a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention and a pharmaceutically acceptable carrier or excipient;

-a vaccine composition comprising a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention or coated particles of the invention.

Also provided is the use of a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention or coated particles of the invention for the preparation of a medicament for nucleic acid vaccination.

These and other subjects, aspects, embodiments, and advantages of the invention will be apparent to those of ordinary skill in the art in view of the disclosure herein.

Drawings

FIG. 1 shows expression levels of hepatitis B virus surface antigen (HBsAg) obtained using various plasmid expression vectors.

FIG. 2 shows the effect of intron addition on HBsAg in SCC15 cells and β -gal expression in B16 cells (average of three experiments).

FIG. 3 shows the effect of rat insulin intron A and HBV 3' UTR on β -gal expression in SCC15 cells (average of three experiments).

FIG. 4 shows the effect of rat insulin intron A and HBV 3' UTR on HSVgD expression in SCC15 cells (average of three experiments).

Figure 5 shows the effect of rat insulin intron a and HBV 3' UTR on SEAP expression in SCC15 and B16 cells (three replicates per cell line).

Figure 6 shows the ability of heterologous signal peptides to direct the secretion of SEAP or hFc fragments in B16 cells.

Figure 7 shows the levels of antibodies detected in the serum of mice immunized with antigen-encoding nucleic acids contained in various plasmid expression vectors.

FIG. 8 is a schematic diagram of a pPJV expression vector.

Fig. 9 is a schematic diagram of pppjv 7389.

FIG. 10 is a schematic diagram of pPJV 7400.

FIG. 11 is a schematic diagram of pPJV 7468.

FIG. 12 is a schematic diagram of pPJV 7563.

FIG. 13 shows the base composition of pPJV 7563.

FIG. 14 provides a flowchart of the derivation of plasmids pPJV7563 and pPJV 1671.

FIG. 15 provides a map of key plasmids in the construction of pPJV 1671.

FIG. 16 provides a feature map of pPJV1671 and provides a sequence comparison of the N-terminal sequence of native H3 Panama HA antigen and the H3 Panama HA antigen encoded by pPJV 1671.

FIG. 17 shows hemagglutination-inhibiting antibody titers in pigs vaccinated with pPJV 1671.

Figure 18 shows the local skin response following delivery of pppjv 1671 to humans in the form of particle-mediated epidermal delivery.

Figure 19 shows hemagglutination inhibition antibody titers in swine immunized with PMED-based trivalent vaccine. HI titers specific for the two PMED devices against H3, H1, and B viruses are shown.

FIG. 20 is a schematic representation of pPML 7789.

FIG. 21 shows the annotated sequence of pPML 7789.

Fig. 22 is a schematic diagram of the pppjv 2012.

FIG. 23 is a flowchart of the construction of pPJV 2012.

Fig. 24 provides an annotated sequence of the pppjv 2012.

FIG. 25 is a schematic diagram of vector pPJV 7788.

FIG. 26 is a schematic diagram of vector pPJV7788 showing restriction sites.

FIG. 27 is a schematic of pICP 27.

FIG. 28 is the amino acid sequence of ICP27 from an HSV-2 MS strain (SEQ ID NO: 65) based on the nucleotide sequence of pICP 27. The single amino acid difference between the published ICP27 sequences of HSV-2 strain MS (asparagine ═ N) and strain HG52 (lysine) is shown in bold. The sequence identified as the putative CD8 epitope in HSV-2 strain MS is underlined.

FIG. 29 shows the identification of the dominant ICP27 epitope in Balb/c mice. A: spleen (S) and lymph node (N) cells (BS and BN, respectively) from HSV-2 infected Balb/C mice and spleen and lymph node cells (CS and CN) from HSV-2 infected C57BL/6 mice were analyzed for IFN- γ ELISPOT activity using the individual peptide pools described in example 22 (C1-C12, R1-R6). Will be 5X 10⁵Individual cells were plated in each well and results were expressed as negative (blank squares), weak response (< 25 ELISPOT/well, grey squares) or strong response (> 25 ELISPOT/well, black squares). B: two peptide sequences recognized by Balb/c mouse cells (SEQ ID NOS: 66 and 67), highlighted are possible HSV-2 Dd epitopes (bold), and a region (underlined) corresponding to the previously identified HSV-1 epitope (Bank et al, J.Virol). 67，613-616，1993)。

FIG. 30 shows characterization of Balb/c response to ICP27 using IFN- γ ELISPOT analysis. The spleen cells of infected Balb/c mice were analyzed for IFN-. gamma.ELISPOT activity using a pool of peptides prepared from the entire peptide of ICP27 (pool of peptides) or individual peptides HGPSLYRTF (P1, SEQ ID NO: 68) and LYRTFAANPRA (P2, SEQ ID NO: 69). Will be 5X 10⁵Individual cells were seeded in each well and the results are expressed as ELISPOT number/well. Additionally, aliquots of the splenocytes samples were treated with magnetic beads to remove CD8+ cells samples as described in example 22. The primary samples (+ CD8) and the treated samples (-CD8) were analyzed for IFN-. gamma.ELISPOT activity using peptide pools (peptide pools) prepared from all peptides of ICP 27.

FIG. 31 shows the correlation between HSV-2 challenge protection and ICP-27 specific cytokine production. Each group of 16 mice had a primary and booster PMED DNA immunization consisting of a plcp 27 DNA vaccine with and without the CT DEI vector pPJV2013 (bis a + B subunit) or the subunit LT DEI vector pPJV2012 (bis a + B). pICP27 and DEI vectorThe ratio of the bodies was 9: 1. FIG. A: survival data after challenge. Half of the animals in each group were treated with 50 LD two weeks after the second immunization ₅₀The HSV-2 of (1) makes a challenge. FIGS. B and C: the production of ICP-27 specific IFN-. gamma.and TNF-. alpha.in each group, respectively. The remaining animals were sacrificed simultaneously and splenocytes were collected for measurement of ICP-27 specific cytokine production using a microsample multi-index flow protein quantification (cytometric bead array) kit.

FIG. 32 shows the protective role of IFN-. gamma.and TNF-. alpha.in the assessment of the mortality challenge-morbidity of HSV-2. 4 mice from each group were primed and boosted with a plcp 27 PMED DNA vaccine with (4 groups) or without (1 group) the pPJV 2012 double a + B LT DEI vector. The ratio of pICP27 to DEI vector was 9: 1. Two weeks later 50 LD was used by intranasal route₅₀HSV-2 challenged animals. To remove T cells, mice were injected intraperitoneally with 200 μ g of an injection solution containing a monoclonal antibody specific for IFN-. gamma.and/or TNF-. alpha.on days-2, 0, 2, 4, 6 and 8 from virus challenge as described in example 22. Animals were monitored for mortality (percent survival) and morbidity and scored according to the following list on a scale of 0 to 4: 4, health; 3, the fur shakes and sneezes; 2, soreness of the eyes or buttocks, decreased mobility; 1, bowing back, almost inactive; 0, death.

FIG. 33 shows the protective effect of T cell populations in a lethal challenge of HSV-2. Five groups of 4 mice per group were primed and boosted with pcipcp 27 PMED DNA vaccine with (+ LT, 4 groups) or without (-LT, 1 group) pPJV2012 double a + BLT DEI vector. The ratio of pICP27 to DEI vector was 9: 1. Two weeks later, 50 LD was used by intranasal route₅₀HSV-2 challenged animals. Three groups of animals immunized with ICP27+ LT DEI were treated by intraperitoneal injection of 90 μ g α CD4 and/or α CD 8-specific monoclonal antibodies on days-2 and 0 from virus challenge. To equalize the total DNA of the pICP 27-only group (denoted-LT) and the pICP27+ LT group, an empty vector was added to the pICP 27-only group.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the hCMV immediate early promoter sequence (GenBank # M60321, X17403)

SEQ ID NO: 2 are sequences of exons 1 and 2 of the hCMV major immediate early gene (GenBank # M60321, X17403)

SEQ ID NO: 3 is the sequence of rat insulin intron A (GenBank # J00748)

SEQ ID NO: 4 is a sequence of the chimeric promoter of the present invention

SEQ ID NO: 5 is leader sequence of HBV preS2 antigen 5' UTR sequence (GenBank # M54923)

SEQ ID NO: 6 is the leader sequence of the HSV type 2gD 5' UTR sequence (GenBank # Z86099)

SEQ ID NO: 7 is leader sequence of 5' UTR sequence of HBV e antigen (GenBank # M54923)

SEQ ID NO: 8 is the sequence of HBVenh 3' UTR (GenBank # AF143308)

SEQ ID NO: 9 is a sequence of the 3' UTR of the simian immediate early gene (GenBank # M16019)

SEQ ID NO: 10 is the polyadenylation sequence of rabbit globin (GenBank # K03256)

SEQ ID NO: 11 is the polyadenylation sequence of the Simian sCMV immediate early Gene (GenBank # M16019)

SEQ ID NO: 12 is the polyadenylation sequence of the HSV2 gB gene (GenBank # Z86099)

SEQ ID NO: 13 is the polyadenylation sequence of the HPV16 early gene (GenBank # K02718).

SEQ ID NO: 14 is the sequence of pPJV expression vector

SEQ ID NO: 15 is a PCR primer JF93

SEQ ID NO: 16 is a PCR primer F110

SEQ ID NO: 17 is PCR primer GW1

SEQ ID NO: 18 is a PCR primer JF254

SEQ ID NO: 19 is PCR primer GW150

SEQ ID NO: 20 is a PCR primer JF255

SEQ ID NO: 21 is PCR primer DS1

SEQ ID NO: 22 is PCR primer DA1

SEQ ID NO: 23 is a PCR primer JF301

SEQ ID NO: 24 is a PCR primer JF302

SEQ ID NO: 25 is a PCR primer JF84

SEQ ID NO: 26 is a PCR primer JF225

SEQ ID NO: 27 is a PCR primer JF335

SEQ ID NO: 28 is a PCR primer JF336

SEQ ID NO: 29 is PCR primer JF357

SEQ ID NO: 30 is a PCR primer JF365

SEQ ID NO: 31 is a PCR primer JF393

SEQ ID NO: 32 is a PCR primer JF406

SEQ ID NO: 33 is a PCR primer JF256

SEQ ID NO: 34 is a PCR primer JF257

SEQ ID NO: 35 is a PCR primer JF320

SEQ ID NO: 36 is a PCR primer JF321

SEQ ID NO: 37 is a PCR primer JF386

SEQ ID NO: 38 is a PCR primer FcAS

SEQ ID NO: 39 is the oligonucleotide JF354

SEQ ID NO: 40 is a PCR primer JF355

SEQ ID NO: 41 is PCR primer JF356

SEQ ID NO: 42 is the oligonucleotide JF348

SEQ ID NO: 43 is a PCR primer JF349

SEQ ID NO: 44 is a PCR primer JF350

SEQ ID NO: 45 is the oligonucleotide JF351

SEQ ID NO: 46 is a PCR primer JF352

SEQ ID NO: 47 is a PCR primer JF353

SEQ ID NO: 48 is a PCR primer JF430

SEQ ID NO: 49 is a PCR primer JF442

SEQ ID NO: 50 is PCR primer JF421

SEQ ID NO: 51 is PCR primer JF444

SEQ ID NO: 52 is a pseudorabies virus (PRV) promoter sequence.

SEQ ID NO: 53 is the Rous Sarcoma Virus (RSV) promoter sequence.

SEQ ID NO: 54 provides the nucleotide sequence of pPJV1671 vector and the amino acid sequence of the encoded H3N2 HA antigen.

SEQ ID NO: the amino acid sequence of the H3N2 HA antigen encoded by pPJV1671 is provided by the disclosure 55.

SEQ ID NO: 56 provides the native N-terminal amino acid sequence of the H3 panama HA antigen.

SEQ ID NO: 57 provides the N-terminal amino acid sequence of the H3 Pananama HA antigen encoded by pPJV 1671.

SEQ ID NO: 58 provides the consensus sequences of sequences SEQ ID Nos. 56 and 57.

SEQ ID NO: 59 provides the nucleotide sequence of the pPML7789 vector and the amino acid sequence of VN1194H5 antigen.

SEQ ID NO: 60 provides only the amino acid sequence of VN1194H5 antigen.

SEQ ID NO: 61 provides the nucleotide sequence of the pPJV2012 vector.

SEQ ID NO: the nucleotide sequence of the PJV7788 vector is provided at 62.

SEQ ID NO: 63 and 64 are the 5 'and 3' primers used in example 22 to amplify DNA from the HSV-2 MS strain.

SEQ ID NO: 65 is the amino acid sequence of ICP27 from the HSV-2 MS strain of example 22 based on the nucleotide sequence of pICP 27.

SEQ ID NO: 66 and 67 are peptides 45 and 46 recognized by the cells of Balb/c mice in example 22.

SEQ ID NO: 68 is a homologous sequence of 9 amino acids contained in peptides 45 and 46.

SEQ ID NO: 69 and 70 are regions of HSV-2 ICP27 and HSV-1 ICP27 which have amino acids which differ by one amino acid.

Detailed Description

Before describing the invention in detail, it should be understood that: the invention is not limited to the particular illustrated molecules or process parameters, as there can, of course, be varied in many ways. It should also be understood that: the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. In addition, the practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology, recombinant DNA techniques, and immunology, all of which are well known to those of ordinary skill in the art. These techniques are fully described in the literature, see, for example, molecular cloning, by Sambrook et al: a laboratory Manual (second edition, 1989) (Sambrook, et al, Molecular Cloning: A laboratory Manual (2nd edition, 1989)), "DNA Cloning: a method of experiment, volumes I and II (DNA Cloning: A Practical Approach, vol. I & II (D.Glover, ed.)), "Oligonucleotide Synthesis (N.Gate ed., 1984))," Molecular Cloning Guide (A Practical Guide to Molecular Cloning (1984)) and "basic Virology second Edition volumes I and II (Fundamental Virology, 2nd Edition, vol. I & II (B.N.fields and D.M.Knipe, eds.)).

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

It must be noted that: as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Where a particular agent is explicitly indicated to comprise a particular unit, the agent may, in preferred cases, consist essentially of such units.

A. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

The following terms will be used in describing the present invention and are defined to have the following meanings.

The term "nucleic acid immunization" as used herein is intended to mean the introduction of a nucleic acid molecule encoding one or more selected antigens or polypeptides into a host cell to effect expression of the one or more antigens in vivo. The nucleic acid molecule can be introduced directly into the recipient subject, e.g., by standard intramuscular or intradermal injection, transdermal particle delivery, inhalation, topical administration, or by oral, intranasal, or mucosal administration. In particular, the nucleic acid may be administered by transdermal particle delivery. Alternatively, the molecule may be introduced into cells removed from the subject in vitro. In the latter case, the cells containing the nucleic acid molecule of interest are reintroduced into the subject, such that an immune response against the antigen encoded by the nucleic acid molecule is initiated. The nucleic acid molecules used in this immunization are generally referred to herein as "nucleic acid vaccines". Any nucleic acid mentioned herein may be present in such a vaccine, in particular, the nucleic acid construct mentioned herein may be present.

The term "adjuvant" refers to a substance or composition that specifically or non-specifically alters, enhances, directs, redirects, enhances or initiates an antigen-specific immune response. Thus, co-administration of the adjuvant and antigen can reduce or diminish the dose of antigen necessary to achieve a desired immune response in a subject to whom the antigen is administered, or alternatively, co-administration can produce qualitatively and/or quantitatively different immune responses in the subject. In particular, administration of the adjuvant can enhance the immune response, such as one of increasing the intensity and/or extending the duration. The effect of the adjuvant was determined by the following method: the adjuvant and vaccine compositions, as well as the vaccine composition itself, are administered to the animals and the antibodies and/or cell-mediated immunity in the two groups are compared by using standard detection methods such as radioimmunoassay, ELISA and CTL assays. The constructs of the invention may express one or more adjuvant polypeptides.

By "core vector" is meant a vector coated with a guest nucleic acid (e.g., DNA, RNA) in order to impart a defined particle size and density high enough to obtain the momentum required to penetrate the cell membrane so that the guest molecule can be delivered using particle-mediated techniques (see, e.g., U.S. patent No.5,100,792). The core carrier typically comprises materials such as tungsten, gold, platinum, ferrite, polystyrene, and latex. See, e.g., Particle bombardment for Gene Transfer (Particle Bombardmenttechnology for Gene Transfer, (1994) Yang, N.ed., Oxford university Press, N.Y., pages 10-11).

By "needleless syringe" is meant an apparatus that delivers the particulate composition transdermally without the aid of a conventional injection needle to pierce the skin. The needleless injector used in the present invention will be described in detail herein.

The term "transdermal" delivery refers to intradermal (e.g., into the dermis or epidermis), transdermal (e.g., "transdermal"), and transmucosal administration, e.g., by injection or delivery of an agent through the skin or mucosal tissue. See, e.g., Transdermal Drug Delivery: development Issues and research initiatives, Hadgraft and Guiy (eds.), Marcel Dekker, Inc. (1989); controlled Drug Delivery: fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc. (1987); and Transdermal Delivery of Drugs, Vols.1-3, Kydonieus and Berner (eds.), CRC Press (1987). Thus, the term includes both needleless syringe delivery (described in U.S. patent No.5,630,796) and particle-mediated delivery (described in U.S. patent No.5,865,796).

"polypeptide" is used in the broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other pseudopeptides (peptidomimetics). The subunits may be linked by peptide bonds or other bonds, such as aliphatic bonds, ether bonds, and the like. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including glycine and both optical isomers in the D or L form, and amino acid analogs and pseudopeptides. If the peptide chain is short, peptides of three or more amino acids are generally referred to as oligopeptides. If the peptide chain is long, the peptide is generally referred to as a polypeptide or protein.

"antigen" refers to any substance, generally a macromolecule, that is capable of eliciting an immune response in an individual. The term may be used to refer to a single macromolecule or to a population of homogeneous or heterogeneous antigenic macromolecules. As used herein, "antigen" is used to generally refer to a protein molecule or portion thereof that comprises one or more epitopes. For example, an "antigen" may comprise a naturally occurring polypeptide, an immunogenic fragment thereof, or a variant of both that retains immunogenicity. In a preferred embodiment, this refers to a fragment or variant, the resulting immune response preferably being capable of recognizing the original polypeptide from which the fragment or variant was derived.

For the purposes of the present invention, an antigen may be obtained or obtained from any suitable source. For the purposes of the present invention, "antigen" includes modified proteins such as deletions, insertions, and substitutions of the native sequence (which are generally conserved in nature) so long as the protein retains sufficient immunogenicity. These modifications may be deliberate, as by site-directed mutagenesis, or accidental, as by mutation of the host producing the antigen. In a particularly preferred embodiment of the invention, the antigen employed or encoded may be an influenza antigen, an immunogenic fragment of an influenza antigen, or an immunogenic variant of both.

An "immune response" to an antigen of interest is the generation of a humoral and/or cellular immune response to the antigen in an individual. For the purposes of the present invention, "humoral immune response" refers to an immune response mediated by antibody molecules, while "cellular immune response" refers to an immune response mediated by T-lymphocytes and/or other leukocytes.

The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length (deoxyribonucleotides or ribonucleotides, or analogs thereof). The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

Polynucleotides generally consist of a specific sequence of four nucleotide bases, adenine (a), cytosine (C), guanine (G), and thymine (T) (uracil (U) in place of thymine (T) when the polynucleotide is RNA). Thus, the term nucleic acid sequence is a letter representation of a polynucleotide molecule. Such alphabetical representations can be entered into a database of a computer having a central processing unit for bioinformatics applications such as functional genomics and homology queries.

"vectors" are capable of transferring a nucleic acid sequence to a target cell, such as viral vectors, non-viral vectors, particulate vectors, and liposomes. The target cell may be a prokaryotic cell or a eukaryotic cell. In general, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest, which can transfer the gene sequence to a target cell. Thus, the term includes cloning and expression vectors as well as viral vectors. A "plasmid" is a vector in the form of an extrachromosomal genetic element.

A nucleic acid sequence "encoding" a selected antigen refers to a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro, under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a 5 'start codon (amino terminus) and a 3' translation stop codon (carboxy terminus). For the purposes of the present invention, these nucleic acid sequences include, but are not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, viral genomic sequences or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

In some cases, the transcribed sequence can produce multiple polypeptides, e.g., a transcript can comprise multiple translatable frameworks (ORFs) and also comprise one or more Internal Ribosome Entry Sites (IRES) such that the ORF after the first ORF can be translated. The transcript may be translated to produce a polypeptide which is subsequently cleaved to form a plurality of polypeptides. In certain instances, a nucleic acid construct can produce multiple transcripts, thereby producing multiple polypeptides.

A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide encoding a polypeptide. Promoters include inducible promoters (inducible by the analyte of expression of a polynucleotide sequence, cofactor, regulatory protein, etc., operably linked to the promoter), repressible promoters (repression by the analyte of expression of a polynucleotide sequence, cofactor, regulatory protein, etc., operably linked to the promoter), and constitutive promoters. The term "promoter" or "regulatory element" includes the full-length promoter regions and functional (e.g., transcriptional or translational regulatory) segments of these regions.

"operably linked" refers to an arrangement of elements wherein the components described in that term are assembled to perform their ordinary function. Thus, a given promoter operably linked to a nucleic acid sequence can affect the expression of that sequence in the presence of the appropriate enzyme. The promoter need not be contiguous with the sequence, so long as it directs expression of the sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and a nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

"recombinant" is used herein to describe a nucleic acid molecule (polynucleotide) of genomic, cDNA, semisynthetic, or synthetic origin which, depending on its origin or method of operation, is not linked to all or part of the polynucleotide to which it is naturally linked and/or to a polynucleotide to which it is not naturally linked. Two nucleic acid sequences in a single recombinant nucleic acid molecule are "heterologous" with respect to each other when they are not normally associated with each other in nature.

Homologs of the polynucleotides are mentioned herein. Typically, a polynucleotide that is homologous to another polynucleotide is at least 70% homologous, preferably at least 75%, 80% or 90%, and more preferably at least 95%, 97% or 99% homologous to the polynucleotide. Methods for detecting homology are well known in the art, and one of ordinary skill in the art will understand that: in this context, homology is calculated on the basis of the identity of nucleic acids. The homology may be present over a region of at least 15, preferably at least 30, such as at least 40, 60 or 100 or more contiguous nucleotides. The homologous region may comprise at least 150, preferably at least 200, more preferably at least 300 nucleotides. Homologous regions may relate to any of the elements mentioned herein in relation to the nucleic acid construct of the invention. In some cases, a homologous region can include all regions to be studied, e.g., can include all regions of any element specifically identified herein.

The level of amino acid homology is comparable to the levels associated with the above nucleotide homology. Thus, any of the above homology levels can be used at the amino acid level. For example, homology at the amino acid level may span at least 15, preferably at least 25, more preferably at least 50, even more preferably at least 75, even more preferably at least 100 amino acids. The homologous regions may span the entire length of the element under study.

Methods for detecting homology or identity of polynucleotides are well known in the art. For example, the UWGCG Package provides the BESTFIT program, which can be used to calculate homology (e.g., using the default settings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395).

The PILEUP and BLAST algorithms can also be used to calculate homology or aligned sequences (typically in default settings), as described in Altschul S.F, (1993) J Mol Evol 36: 290-300; altschul, S, F et al (1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available through the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). The algorithm first identifies high-ratio sequence pairs (HSPs) by identifying short fields of length W in the sequence to be queried that match or satisfy some positive-valued threshold score T when aligned with a field of the same length in a database sequence. T refers to the neighbor score threshold (Altschul et al, supra). These initial neighbor field hits act as seeds for starting a search to find HSPs containing them. The field hits extend in both directions along each sequence, maximizing the cumulative alignment score. The following situation occurs, namely the extension of the stall field hit in both directions: when the cumulative alignment score is zero or less than zero, this is due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. BLAST program uses default values: the field length (W) was 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences, see, e.g., Karlin and Altschul (1993) proc.natl, acad.sci.usa 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)). it provides an indication of the probability that a match will occur randomly between two nucleotide or amino acid sequences. For example, a first sequence is considered similar to a second sequence if the smallest sum probability when comparing the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Homologs typically hybridize to the relevant polynucleotide at levels significantly above background. The signal level resulting from the interaction between the homologue and the polynucleotide is generally at least 10-fold, preferably at least 100-fold that of the "background hybridization". The strength of the interaction may be determined using, for example, radiolabels such as³²P is detected by a probe. Selective hybridization is generally achieved by using moderate to high stringency conditions (e.g., about 50 ℃ to about 60 ℃, 0.03M sodium chloride and 0.003M sodium citrate).

Stringent hybridization conditions may include 50% formamide, 5 XDenhardt's solution, 5 XSSC, 0.1% SDS and 100. mu.g/ml denatured salmon sperm DNA, and washing conditions may include a 2 XSSC, 0.1% SDS wash at 37 ℃ followed by a 1 XSSC, 0.1% SDS wash at 68 ℃. The determination of suitable hybridization conditions is well known to those of ordinary skill in the art, see, e.g., Sambrook, et al, supra.

Homologs may differ from a sequence by fewer than 3, 5, 10, 15, 20, or more mutations (each of which may be a substitution, duplication, deletion, or insertion) in the relevant polynucleotide. These mutations may be determined over a region of at least 30, such as at least 40, 60 or 100 or more contiguous nucleotides of the homologue. In some cases, mutations can be measured over the entire region of the homolog. Where the polynucleotide encodes a polypeptide, preferably, the substitution results in a "conservative" change in the encoded amino acid. Conservative changes are defined according to the following table. In conservative variations, amino acids in the same square of the second column, preferably in the same row of the third column, are substituted for one another.

TABLE 1

Aliphatic series	Non-polar	GAP
		GAP	ILV
		Polar-uncharged	ILV	CSTM
	NQ			CSTM
	NQ		Polar-charged	DE
	KR			DE
	KR	Aromatic compounds			HFWY

The term "fragment" refers to a smaller portion of a larger entity. Fragments of the specific elements mentioned herein may be used in the present invention. In particular, these fragments retain some or all of the functionality of the original element, particularly any of the functionality mentioned herein. In a preferred embodiment, a fragment of the antigen may retain immunogenicity, and a fragment of the adjuvant retains the ability to act as an adjuvant. In some cases, the length of a fragment is at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 80%, even more preferably at least 90%, still more preferably at least 95% of the original element. The segments may be equal to or less than these percentages of the original element length.

The terms "individual" and "subject" are used interchangeably herein to refer to any member of the vertebrate subgenus, including, but not limited to, humans and other primates, including primates other than humans such as chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals include rodents such as mice, rats and guinea pigs, and pigs; birds, including domesticated birds, wild birds and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, geese, and the like. The term does not refer to a particular age. Thus, both adult and newborn individuals are included. Since the immune system of all of these vertebrates functions in a similar manner, the methods described herein are useful for any of the vertebrate species described above.

In certain instances, the present invention may be used to immunize any suitable subject, particularly any suitable subject of a given species. In a preferred embodiment, any suitable human subject may be immunized. Thus, for example, as many subjects as possible can be immunized without having to emphasize subjects of any particular cohort. For example, the immunization may be performed on a population of subjects as a whole or as many as possible. In particular, when the invention is used to treat influenza, particularly to immunize against an epidemic influenza virus strain, the constructs of the invention may be used to immunize any subject and preferably as many subjects as possible.

In other cases, the subject or individual may be one who is at risk of infection or for whom infection is particularly detrimental. In a preferred example, the infection may be a respiratory infection. In particular, when the present invention is used to prevent or treat respiratory infections, particularly influenza, the subject may be a human. In certain instances, the nucleic acid constructs of the invention may be administered preferentially or first to a particular subject of the risk group. For example, this may be the case: the nucleic acid construct is administered to immunize against a non-influenza virus strain. For example, in certain instances, subjects may be classified into one or more of the following categories:

-a subject having a respiratory disorder and/or cardiac problem, in particular a subject suffering from asthma, emphysema, bronchitis and/or Chronic Obstructive Pulmonary Disease (COPD);

-subjects suffering from chronic medical conditions such as diabetes, immunosuppression, immunodeficiency, sickle cell disease and/or renal disease;

-a subject at least 50 years of age, preferably at least 60 years of age, more preferably at least 65 years of age, even more preferably at least 75 years of age, yet more preferably at least 80 years of age;

children aged 2 or less than 2 years, in particular children aged 6 to 23 months, for example 18 months or less than 18 months;

-subjects on chronic aspirin treatment, in particular subjects aged 6 months to 18 years;

-pregnant women, in particular pregnant women which will be in the middle or third trimester of pregnancy during the flu season;

residents of the retirement home or residents of the long-term care institution; and/or

The caregivers of any of the above groups or the persons with whom they may be in frequent contact.

B. Overview

The present invention relates to nucleic acid constructs which provide the possibility of efficient expression of heterologous coding sequences, particularly antigen-encoding genes, in host cells. In certain instances, the construct may express one or more adjuvant polypeptides. More specifically, the invention provides nucleic acid constructs comprising, or in certain embodiments consisting essentially of, a chimeric promoter sequence and a cloning site, such that when the coding sequence is inserted into the cloning site, the coding sequence is operably linked to the chimeric promoter. The invention also provides constructs having coding sequences inserted into one or more cloning sites. The coding sequence may encode any of the polypeptides mentioned herein, particularly antigens, in particular any of the antigens and adjuvant polypeptides described herein.

In a particularly preferred embodiment, the construct comprises a coding sequence, in particular a coding sequence encoding an immunogenic variant of the antigen, an immunogenic fragment thereof or both. In a particularly preferred embodiment, the coding sequence encodes an immunogenic variant of an influenza antigen, an immunogenic fragment thereof, or both. In a more preferred embodiment, the coding sequence encodes an adjuvant polypeptide, in particular an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity or a variant thereof in which both have adjuvant activity.

Chimeric promoters comprise, or in certain embodiments consist essentially of, the following elements:

(a) hCMV immediate early promoter sequence;

The hCMV immediate early promoter sequence (a) may comprise:

(i) a native hCMV immediate early promoter sequence;

(ii) a functionally homologous variant thereof; or

(iii) (iii) a functional fragment of (i) or (ii).

Generally, sequence (a) comprises about 100 to 600, preferably 200 to 600, for example 400 to 600 nucleotides. Sequence (a) generally includes, but is not limited to, sequences present in (i) that bind to a transcription factor or RNA polymerase, or homologues of such sequences that bind to the same transcription factor and RNA polymerase. Typically, these sequences or homologues thereof are present in the promoter sequence (a) in the same order and/or at substantially the same relative spacing as (i).

Typically, (i) includes nucleotides at least the-100 to-1, typically-150 to-1, positions, such as-500 to-1 or-600 to-1, positions of the major immediate early gene of hCMV. Sequence (i) typically comprises the hCMV core promoter sequence and may also comprise one or more enhancer elements present in the hCMV immediate early promoter. For example, (i) may comprise a nucleotide from position-118 to-1 or from position-524 to-1 as described in US 6218140 or a nucleotide from position-62 to-1 or from position-465 to-1 as described in US 5385839.

Typically, (i) includes a TATA box or CAAT box typically found in promoter sequences. Preferably, the sequence comprises one or more repeats in the hCMV immediate early promoter.

In a preferred embodiment, (i) comprises SEQ ID NO: 1. in a more preferred embodiment, (i) comprises SEQ ID NO: nucleotide 903 to 1587 of 54. In another embodiment, (i) may comprise SEQ ID NO: nucleotides 903 to 1587 of 14. In a more preferred embodiment, (i) may comprise nucleotides 1002 to 1686 or 2624 to 3308 of SEQ ID No. 57. In a more preferred embodiment, (i) may comprise nucleotides 1815 to 1935 and/or 1948 to 2632 of SEQ ID No.61, in particular nucleotides 1948 to 2632 of SEQ ID No. 61. In another preferred embodiment, nucleotides 1815 to 1935 may be used.

In a particularly preferred embodiment of the invention, the hCMV immediate early promoter sequence (a) comprises:

(i) nucleotide sequence SEQ ID No.1, nucleotides 903 to 1587 of SEQ ID No.54, SEQ ID No: nucleotide 1815 to 1935 of 61, SEQ ID No: 61 from nucleotide 1948 to nucleotide 2632, SEQ ID No: 62 from nucleotide 1002 to 1686 and/or SEQ ID No: nucleotides 2624 to 3308 of 62;

(ii) (ii) a functional variant of (i) having at least 80% nucleotide sequence homology to one or more sequences of (i); and/or

(iii) (iii) a functional fragment of (i) or (ii).

In certain instances, a fragment may comprise at least 300 nucleotides, preferably at least 400 nucleotides, more preferably at least 500 nucleotides, even more preferably at least 600 nucleotides of the sequence. Fragments may comprise up to 800, up to 600, or up to 400 nucleotides.

The hCMV immediate early promoter sequence can be obtained using known methods. The native hCMV immediate early promoter can be isolated directly from viral samples using standard techniques. For example US 5385839 describes the cloning of the hCMV promoter region. The hCMV immediate early promoter sequence is available from Genbank # M60321(hCMVTowne strain) and X17403(hCMV Ad169 strain). Thus, the native sequence can be isolated by PCR using PCR primers based on known sequences. See, e.g., Sambrook et al (supra) for a description of techniques for obtaining and isolating DNA. Suitable hCMV promoter sequences can also be isolated from existing plasmid vectors. Promoter sequences may also be produced synthetically.

The functional variant (ii) or fragment (iii) generally maintains and/or complements the activity of the native promoter (i). Generally, such activity refers to the ability to cause (including initiate and regulate) transcription of an operably linked polynucleotide, particularly the hCMV major immediate early gene. In one embodiment, the fragment or variant is capable of complementing the activity of a native promoter in an hCMV virus, such as to allow the virus to maintain the ability to infect and/or replicate within a cell.

The ability of the homologous variant (ii) or fragment (iii) to maintain and/or complement the activity of (i) can be detected. For example, the ability of a variant or fragment to restore function (e.g., infection and/or replication capacity) to a mutant hCMV in which the native hCMV immediate early promoter is defective can be detected.

The homologous variant (ii) or fragment (iii) can be tested for availability using differential expression analysis (comparative expression Assay) as follows. The test promoter sequence was introduced into the basic vector (basevector) replacing the native hCMV immediate early promoter. Generally, functional variants or fragments can achieve at least 50%, such as 60%, 70%, 80%, 90% or more of the expression provided using the base vector. Functional variants or fragments may provide for any level of expression described herein. In certain instances, a variant or fragment may provide higher levels of expression, e.g., an increase in activity of at least 50%, 100%, 150%, 200%, 300%, or more. Typically, expression occurs in at least one, but preferably two, reference cell types. Typically, the reference cell is a mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cell. In some cases, the reference cell may be an SSC-15 or B16 host cell.

Additionally or alternatively, the promoter sequence may be detected by the following differential Immunogenicity Assay (Comparative Immunogenicity Assay). The test promoter sequence was introduced into the basic vector, replacing the native hCMV immediate early promoter. In general, the functional promoter sequence provides an antibody titer which is at least the same as or higher than the antibody titer obtained with the basic vector having at least one antigen, preferably two antigens. Preferably, the antibody titer is at least 5%, 10%, 20%, 30% or 40% higher than that of the base vector. In certain instances, the level of antigen titer can be any level described herein. Suitable antigens are HBsAg, HSV 2gD and flu-M2 antigens. Particularly preferred antigens are influenza antigens. Influenza antigens include HA, NA, M2, NP, M1, PB1, PB2, PA, NS1 and NS2 antigens, in particular HA, NA and M2 antigens. In a particularly preferred embodiment, the antigen is HA or a fragment thereof, or a variant of both. According to the present assay, the highest antibody titers obtained with the native sequence are generally obtained with functionally homologous variants (ii) or functional fragments (iii) of the native promoter sequence (i). In certain instances, the antibody titer may be slightly lower, including any of the levels described herein.

Where the constructs of the invention encode adjuvant polypeptides, a differential immunogenicity test using standard antigens may be employed and the test vector encoding the polypeptide with unknown adjuvant activity compared to standard adjuvant vectors. For example, the test adjuvant vector may encode a fragment or variant of a known adjuvant polypeptide and compare its ability to promote an immune response when administered concurrently with an antigen to a standard vector expressing the known adjuvant polypeptide. The fragment or variant may have any level of activity as described herein, and in particular may provide at least 50%, preferably at least 75%, more preferably at least 85%, even more preferably at least 100% of the adjuvant activity of a standard adjuvant. Preferably, the test vector and the standard vector are identical, or at least substantially identical, except for the sequence encoding the adjuvant/polypeptide to be tested.

As described above, the construct may comprise exon sequences (b) which include sequences derived from exon 1 and exon 2 of the hCMV major immediate early gene. Exons are coding sequences, which are normally separated in nature by introns. In the natural hCMV major immediate early gene, exons 1 and 2 are typically separated by a natural intron a. In the chimeric constructs of the invention, the exon 2 sequence is typically located 3' to the exon 1 sequence, with no intron sequence spacing, so that the exon 1 and exon 2 sequences are contiguous.

Exon sequences (b) may include:

(i) natural exon sequences, typically exon 1 and (all or part of) exon 2;

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

Sequence (i) may comprise about 50% to 100% of the native hCMV major immediate early gene exon 1 sequence, e.g., 60% to 90%, or 70% to 80%. Typically there is at least 50% of the native exon 1 sequence, e.g., 60%, 70%, 80%, 90% or more. Exon sequences (b) also include at least part of exon 2 sequences. In sequence (i), there are typically two or more bases, e.g., 2 to 9, 2 to 7, or 3 to 5 bases, of native exon 2. There is up to and including 100% of the native exon 2 sequence, such as 5% to 95%, 20% to 80%, or 40% to 60% of the native exon 2 sequence. In general, homologous variants have any of the above-described lengths of the native sequence.

Preferably, (i) comprises SEQ ID No. 2. In a more preferred embodiment, (i) comprises SEQ ID NO: nucleotide 1588 to 1718 of 54. In a more preferred embodiment, (i) comprises SEQ ID NO: 14 from nucleotide 1588 to nucleotide 1718. In another preferred embodiment, (i) a polypeptide comprising SEQ ID NO: 51 from nucleotide 1684 to 1814 and/or from nucleotide 2633 to 2763. In a more preferred embodiment, (i) may comprise the amino acid sequence of SEQ ID No: nucleotides 1687 to 1817 and/or 3309 to 3439 of SEQ ID No. 62.

Thus, in a particularly preferred embodiment, the exon sequences (b) of the chimeric promoter comprise:

(i) a nucleotide sequence of the nucleotide sequence SEQ ID No.2, nucleotides 1588 to 1718 of SEQ ID No.54, nucleotides 1684 to 1814 of SEQ ID No.61, nucleotides 2633 to 2763 of SEQ ID No.61, nucleotides 1687 to 1817 of SEQ ID No.62 and/or nucleotides 3309 to 3439 of SEQ ID No. 62;

(iii) (iii) a functional fragment of (i) or (ii).

Suitable exon sequences (b) can be obtained by known methods. See, e.g., Sambrook et al, supra, for a description of techniques for obtaining and isolating DNA. The native hCMV major immediate early gene sequence can be isolated directly from the Virus sample by using standard techniques (see, e.g., Maclean, A (1998) "Preparation of HSV-DNA and Production of Infectious Virus" in Herpes Simplex Virus Protocols (Preparation of Virus-infecting HSV-DNA and products in the Herpes Simplex Virus protocol) ") S.Brown, A Maclean, editors, Humana Press, Totowa, NJ, pp.19-26). The sequence of the hCMV major immediate early gene, including the positions of exon 1 and exon 2, is available in Genbank # M60321, X17403. Thus, the native exon 1 and 2 sequences can be isolated by either cleaving the native major gene sequence at appropriate restriction sites, or by PCR using PCR primers based on known sequences. Alternatively, suitable exonic sequences may be isolated from existing plasmid vectors such as pWRG 7128. Exon sequences may also be prepared synthetically rather than by cloning. Variant sequences are readily constructed by conventional methods, such as site-directed mutagenesis.

In general, when an exon is present in the construct of the invention, it enhances expression, typically resulting in an increase comparable to the native exon 1 and exon 2 sequences (i) described above.

The function of the exon sequences can be analyzed using the differential expression assay described below. The exon sequence to be tested is introduced into the basic vector to replace the existing exon sequence. In general, an exon sequence is functional if its expression is not halted, preferably further increased, in at least one, preferably both, reference cell types compared to the base vector. Typically, the reference cell is a mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cell. SSC15 or B16 cells can be used. Preferably, expression is increased by at least 5%, 10%, 20%, 30% or 40%. Expression may be increased at any level described herein. According to the present assay, a functionally homologous variant (ii) or functional fragment (iii) of a native exon sequence (i) will typically achieve an expression increase of at least 50% compared to the native sequence. In some cases, if the expression level is lower than the basal level, the degree of reduction may be any of the levels described herein.

Additionally or alternatively, exon sequences can be detected using the following differential immunogenicity assay. The exon sequence to be tested is introduced into the basic vector to replace the existing exon sequence. In general, functional exon sequences provide antibody titers that are at least the same as or higher than those obtained for the basic vector having at least one antigen, preferably two antigens. Preferably, the antibody titer is at least 5%, 10%, 20%, 30% or 40% higher than that of the base vector. The degree of elevation may be any level described herein. Preferred antigens are HBsAg, HSV 2gD and flu-M2 antigens. Particularly preferred antigens are influenza antigens, including any of those mentioned above, especially the HA, NA and M2 antigens. It is particularly preferred to use immunogenic variants of the HA antigen, immunogenic fragments thereof or both. According to the present assay, the highest antibody titers obtained with the native sequence are generally obtained with functionally homologous variants (ii) or functional fragments (iii) of the native exon sequence (i). In the case where the level of antibody titer is slightly reduced, the degree of reduction may be any of the levels described herein. The adjuvant vectors can be evaluated in the equivalent forms described above.

The chimeric promoter construct comprises a heterologous intron (c) replacing the native intron a region of the hCMV major immediate early gene. Introns are non-coding sequences resulting from splicing of hnRNA resulting from gene transcription. A heterologous intron is an intron that is not naturally present in the coding sequence.

The heterologous intron (c) replaces, in whole or in part, the natural intron (a) of the hCMV major immediate early gene. Typically, the natural intron a is not present.

Typically, the heterologous intron (c) is located 3' to the exon sequence (b).

Generally, the heterologous intron (c) comprises:

(i) a natural intron;

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

The heterologous intron (c) is typically an intron of a virus or eukaryote. Preferably, the intron is a mammalian intron, particularly an intron other than human, for example, a rat intron may be used. In some cases, chicken introns may also be used. Preferably, the intron is intron a, e.g., rat insulin intron a, chicken keratin intron a, or chicken heart actin intron a. In a particularly preferred embodiment, the intron is rat insulin intron a.

In a preferred embodiment, the heterologous intron (c) comprises a sequence selected from the group consisting of: a rat insulin gene intron a sequence, a chicken keratin gene intron a sequence, a chicken heart actin gene intron a sequence, a functional fragment thereof, or a functional fragment of any of the foregoing.

Typically, intron (c) is from about 50 nucleotides to about 1000 nucleotides in length, such as from about 100 to about 500 nucleotides. For example, intron (c) may comprise 50 to 500 nucleotides, such as up to 100, 200, 300 or 400 nucleotides. Preferably, the intron comprises nucleotides 50 to 133 of intron a of native rat insulin, or a homologue of this sequence.

Preferably, the heterologous intron (c) is capable of splicing from an RNA transcript of a eukaryotic host cell. Generally, introns include one or more of donor sequences (e.g., GT), acceptor sequences (e.g., AG), 3' pyrimidine-rich regions, and branch point sequences. If a pyrimidine-rich region is present, the region may include, for example, at least 5, 8, 10, or more pyrimidines. Preferably, the intron includes at least one donor sequence, one acceptor sequence and one branch point sequence. Typically, in chimeric constructs, intron (c) includes non-intronic flanking sequences derived from exon sequences found at the intron/exon boundaries of the native intron (i). The flanking exon sequences may be native exon sequences or homologues of such sequences which retain substantially the same activity as the native sequence, e.g. retain splicing function. Typically, 5 to 10, preferably 7 to 10 bases of the exon sequence are included at both ends of the intron.

The intron (c) may be an artificial intron, provided that the intron is functional. For example, recombinant introns or chimeric introns may be used. Such introns may include sequences from more than one native intron.

Generally, intron (c) includes sequences present in hCMV intron a that bind to transcription factors or regulatory proteins, or homologues of these sequences capable of binding to the same transcription factors or proteins, rather than these sequences. Typically, these sequences or homologues thereof will be present in intron (c) in the same order and/or at substantially the same relative spacing as in intron a of hCMV.

Intron (c) includes homologous variants (ii) in which the sequence of the native intron (i) is modified to remove internal restriction sites. For example, homologous variants of rat insulin intron A may be used, in which the Nhel site has been disrupted.

Preferably, the intron (c) comprises:

(i)SEQ ID No.3；

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

In a more preferred example, (i) can include SEQ ID NO: 54 from nucleotide 1725 to 1857 or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof. In a more preferred embodiment, (i) may comprise SEQ ID NO: nucleotides 1545 to 1677 and/or 2770 to 2902 of 61. In another preferred example, (i) may comprise SEQ ID NO: nucleotides 1824 to 1956 and/or 3446 to 3578 of 62.

In a preferred embodiment, the rat insulin gene intron a sequence comprises:

(i) nucleotide sequence SEQ ID NO: 3. nucleotide sequence 1725 to 1857 of SEQ ID No.54, SEQ ID No: nucleotide 1545 to 1677 of 61, SEQ ID No: nucleotide 2770 to 2902 of 61, SEQ ID No: nucleotide 1824 to 1956 of 62 and/or SEQ id no: nucleotides 3446 to 3578 of 62;

(iii) (iii) a functional fragment of (i) or (ii).

The intron sequence (c) can be obtained using standard cloning techniques. For example, rat insulin intron a sequences are available in GenBank J00748, chicken keratin intron a sequences are available in GenBank J00847, and chicken heart intron a sequences are available in GenBank X02212. Intron sequences can be isolated from natural sources using primers based on known sequences. Sequences may also be prepared synthetically. Variant sequences may be obtained by mutation.

Typically, a functional intron sequence, such as functional variant (ii) or functional fragment (iii), has substantially the same activity as, and/or complements the activity of, native intron (i). In one embodiment, the activity refers to splicing activity.

The splicing activity of the intron (c) sequence can be detected using conventional splicing assays. Typically, in this assay, functional homologue (ii) or functional fragment (iii) exhibits a splicing efficiency of at least 50%, such as 60%, 70%, 80%, 90% and up to 100% or more of native intron (i).

In general, a heterologous intron sequence, when present in a construct of the invention, enhances expression. In general, the intron of variant (ii) or fragment (iii) is capable of producing a comparable increase as compared to the native intron (i). In some cases, the degree to which expression can be observed to be reduced includes any level recited herein.

The function of the potential intron sequence (c) can be detected using the following differential expression analysis. Heterologous introns are introduced into the basic vector. In general, a heterologous intron sequence is functional if its introduction increases its expression by 25% or more in at least one, preferably both, reference cell types when compared to the basic vector. Typically, the reference cell is a mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cell. SSC15 or B16 cells can also be used. Expression is increased by at least 35%, 45%, 55% or more. The degree of elevation may be any level described herein. According to the present assay, the functional variant (i) or functional fragment (ii) of the native intron sequence (i) is typically expressed more than 50% higher than the native sequence. For example, where decreased expression is observed, the degree of decrease can be any level recited herein.

Additionally or alternatively, the functionality of the heterologous intron (c) sequence can be detected using the following differential immunogenicity assay. The intron (c) sequence is introduced into the base vector. The functional intron (c) sequence provides an antibody titer which is the same as or higher than the antibody titer obtained with the basic vector having at least one, preferably two antigens. Preferably, the antibody titer is increased by at least 5% or 10%, such as 20%, 30% or 40% over the basic vector. Preferred antigens are HBsAg, HSV2gD and flu-M2 antigen. Particularly preferred antigens are influenza antigens, including any of the influenza antigens mentioned herein, in particular the HA, NA and M2 antigens. In particular, immunogenic variants of the HA antigen, immunogenic fragments thereof, or both may be used. According to the present assay, the functional variant (ii) or functional fragment (iii) of the native intron sequence (i) generally achieves the highest antibody titers obtained with the native sequence. For example, where a decrease is observed, the level can be any of the levels recited herein. The adjuvant carrier can be evaluated in the same manner as described elsewhere herein.

Suitable heterologous intron sequences can be obtained using standard cloning techniques. For example, rat insulin intron a sequences are available in GenBank J00748, chicken keratin intron a in GenBank J00847, and chicken heart actin intron a sequences are available in X02212. Intron sequences can be isolated from natural sources using primers based on known sequence data. Suitable sequences may also be prepared synthetically.

The component sequences (a), (b) and (c) may be suitably joined together to form a chimeric promoter using standard cloning or molecular biology techniques. Preferably, the intron sequence (c) is located 3' to the exon sequence (b). The chimeric promoter construct is linked to a cloning site in such a way that the promoter affects the expression of the coding sequence inserted into the site in the presence of a suitable enzyme. Suitable cloning sites, including multiple cloning sites, are well known in the art, such as pUC19, pBC SK, pBluescript IIKS, cDNA3.1, pSP72, pGEM 7Z multiple cloning sites. For example, any suitable restriction enzyme site may be used as a cloning site for inserting the coding sequence.

In a preferred embodiment, the chimeric promoter used comprises:

(i) nucleotides 903 to 1857 of the nucleotide sequence SEQ ID No.4 or the nucleotide sequence SEQ ID No. 54;

(ii) (ii) a functional variant of (i) having at least 80% nucleotide sequence homology to (i); and/or

(iii) (iii) a functional fragment of (i) or (ii).

In a more preferred example, the chimeric promoter used may be a promoter comprising the sequence SEQ ID No: 14 in the presence of a promoter.

Generally, the nucleic acid used to insert (or have inserted) the cloning site encodes a polypeptide that is therapeutically relevant. Preferably the coding sequences are suitable for use in nucleic acid vaccination or gene therapy. Thus, the nucleic acid insert may include a sequence that provides immunity, e.g., an immunogenic sequence, that elicits a humoral and/or cellular immune response when delivered to a subject. Alternatively, the nucleic acid may include one or more genes encoding therapeutic polypeptides, such as proteins defective or absent from the genome of the target cell, or non-native proteins having a desired biological or therapeutic effect, such as antiviral function. The construct may encode an adjuvant polypeptide. For the treatment of genetic diseases, functional genes corresponding to genes known to be deleted in a particular disorder can be administered to a subject. Preferably, the nucleic acid is DNA. In some cases, the nucleic acid may be RNA or PNA.

In certain instances, non-coding RNAs may be expressed by constructs of the invention. Thus, the vector may be inserted into a cloning site, a region that produces such an RNA (e.g., antisense RNA or SiRNA). For example, antisense RNA or SiRNA can inhibit the expression of any one of the genes described herein or of any pathogen described herein. However, in the most preferred embodiment, they encode a polypeptide.

Suitable inserted nucleic acids include those useful for the treatment of inflammatory, autoimmune, chronic, and infectious diseases, including conditions such as aids, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia (hypercholesteremia); various hematological disorders including various anemias (anemias), thalassemia (thalassemia), and hemophilia (hemophilia); genetic defects such as cystic fibrosis (cystic fibrosis), Gaucher's disease, Adenosine Deaminase (ADA) deficiency (adenosine deaminase deficiency), emphysema (emphysema), and the like.

The constructs of the invention may be used to treat or prevent disorders. The constructs may be used to ameliorate a condition and/or eliminate or reduce specific or all symptoms of a disease. In a particularly preferred embodiment, the construct may be used to immunize a subject against a pathogen.

For example, in a method of treating a solid tumor (solid tumor), the following genes may be inserted and expressed at or near the tumor site: genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diphtheria toxin, and cobra venom factor), tumor suppressor genes such as p53, genes encoding mRNA sequences that transduce the antisense strand of the oncogene, genes encoding anti-tumor peptides such as Tumor Necrosis Factor (TNF) and other cytokines, or transdominant negative mutants of the oncogene.

In a preferred embodiment, the construct of the invention may encode a polypeptide for the treatment or prevention of cancer. In a particularly preferred embodiment, the construct of the invention may encode a tumor antigen. Examples of tumor-associated antigens include, but are not limited to, tumor-testis antigens such as members of the MAGE family (MAGE 1, 2, 3, etc.), NY-ESO-1 and SSX-2; differentiation antigens such as tyrosinase, gp100, PSA, Her-2 and CEA; mutated autoantigens and viral tumor antigens such as E6 and/or E7 from oncogenic HPV types. Other examples of specific tumor antigens include MART-1, Melan-A, P97, β -HCG, GaINAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3, MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, K19, Tyr1, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secreted protein, alpha fetoprotein, CA125, CA19.9, TAG-72, BRCA-1, and BRCA-2 antigens.

Examples of specific cancers of antigenic origin include lung cancer, prostate cancer, breast cancer, colon cancer, ovarian cancer, testicular cancer, large bowel cancer, melanoma, lymphoma and leukemia. The constructs of the invention may also be used to treat or prevent such cancers.

Also included are nucleic acids encoding polypeptides known to exhibit antiviral and/or antibacterial activity, or to stimulate the host immune system. The nucleic acid may encode one of a variety of cytokines (or functional fragments thereof), such as interleukins, interferons, and colony stimulating factors (colony stimulating factors).

In a preferred example, the coding sequence may encode an immunogenic variant of an antigen, an immunogenic fragment thereof, or both. In particular, the antigen may be a viral, bacterial, parasitic or fungal pathogen antigen or a tumor antigen. In a preferred example, the antigen may be a viral antigen, an immunogenic fragment thereof, or an immunogenic variant of both.

The nucleic acid may encode an antigen for the treatment or prevention of a number of diseases including, but not limited to, cancer, allergy, intoxication and infection by pathogens such as, but not limited to, fungi; viruses, including Human Papilloma Virus (HPV), HIV, HSV2/HSV1, Influenza Virus (A, B and C), Poliovirus (Poliovirus), RSV Virus (RSVvir), Rhinovirus (Rhinovirus), Rotavirus (Rotavirus), hepatitis A Virus (hepatitis Avirus), Norwalk Virus Group (Norwalk Virus Group), Enterovirus (Enterovirus), Astrovirus (Astrovirus), Measles Virus (Measles Virus), parainfluenza Virus (Para Influenza Virus), Mumps Virus (Mumps Virus), Varicella-zoster Virus (Varicella-zoster Virus), cytomegalovirus (cymegavirus), Epstein-Barr Virus (Virus-Virus), Epstein-Barr Virus (Virus-Barr Virus), Adenovirus-Barr Virus (Adriavirus), Human Lymphoma Virus (LV-cell type LV-T-Virus), Adenovirus (HIV-T-cell, HIV-Virus (HIV-T-cell), HIV 2/HSV1, Rotavirus (Rotavirus), hepatitis A Virus (hepatitis A, hepatitis A Virus), Mumps Virus (Virus), Mumps Virus (Rubus Virus (Mumps Virus), Mumps Virus (Virus), cytomegalovirus (Virus), Human Virus (HIV-T-Virus), Human Lymphoma Virus (HIV-T-Virus, HPV-T-cell type IV-T-Virus (HPV, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D virus (Hepatitis D virus), poxvirus (Pox virus), mare virus and Ebola virus (Marburg and Ebola); a bacterium, comprising: tuberculosis (m.tuberculosis), Chlamydia (Chlamydia), gonorrhea (n.gonorrhoeae), Shigella (Shigella), Salmonella (Salmonella), Vibrio cholerae (Vibrio Cholera), Treponema pallidum (Treponema pallidum), Pseudomonas (Pseudomonas), Bordetella Pertussis (Bordetella Pertussis), Brucella (Brucella), Franciscella herna (Franciscella tubularis), Helicobacter pylori (Helicobacter pylori), leptospira interrogans (leptospora), legionella pneumophila (legionnella), Yersinia pestis (Yersinia pestis), Streptococcus (Streptococcus pneumoniae) (type a and B), actinococcus (Pneumococcus), rhodococcus (rhodobacter), haemophilus (trichomonas), Streptococcus bovis (Streptococcus pneumoniae), mucosae (mucosae), rhodobacter xylinus (trichomonas), and rhodobacter xylinus (trichomonas), trichomonas (trichomonas), Streptococcus bovis (trichomonas pneumoniae); fungal pathogens, including candida (Candidiasis) and aspergillus (aspergillus); parasitic pathogens include tapeworm (Taenia), Flukes (Flukes), roundworms (roundworms), amoebas (Amebiasis), giardia (Giardiasis), Cryptosporidium (Cryptosporidium), schistosoma (Schitosoma), Pneumocystis carinii (pnemocystis carinii), trichomonas repens (trichomonas) and Trichinosis (Trichinosis). The nucleic acids may also be used to provide a suitable immune response against a variety of livestock diseases such as Foot and Mouth Disease (Foot and mouse Disease), Coronavirus (Coronavirus), Pasteurella multocida (Pasteurella multocida), Helicobacter (Helicobacter), strongyloides vulgare (strongyloides), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), bovine viral diarrhea virus (Boyine viral diarrhea virus, BVDV), pneumobacillus (pneumobacillus pneumoniae), escherichia coli (e.coli), Bordetella pertussis (borderella pertussis), Bordetella parapertussis (borderella pertussis paraguas) and Bordetella bronchiseptica (borderella pneumoniae). Thus, in one aspect, the nucleic acid constructs of the invention may be used in vaccines. The vaccines of the present invention can be used to immunize against any of the pathogens and diseases described herein. Accordingly, the present invention provides a vaccine composition comprising a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention or a coated particle of the invention.

In a preferred embodiment, the nucleic acid construct of the invention may encode an antigen from a member of the following virus families: adenoviridae (including, for example, human adenoviruses), Herpesviridae (herpesviridae) (including, for example, HSV-1, HSV-2, EBV, CMV and VZV), papovaviridae (papovaviridae) (including, for example, HPV), poxviridae (poxviridae) (including, for example, smallpox and vaccinia), parvoviridae (paroviridae) (including, for example, parvovirus B19), reoviridae (reoviridae) (including, for example, rotavirus), coronaviridae (coronaviridae) (including, for example, SARS), flaviviridae (flaviviridae) (including, for example, yellow fever virus, West Nile river virus, dengue virus, hepatitis C virus and tick-borne encephalitis virus), picornaviridae (picornaviridae) (including, rhinovirus and hepatitis A), togaviridae (togaviridae) (including, herpesviridae) (including, for example, rubella (mare), and filoviridae) (including, filovirus (marburg) (including, and filovirus) (including, aeviridae) (including, papova), papova (papova), papova) (including, papova), papovaviridae (papova) (including, papova), papova, Paramyxoviridae (including, for example, parainfluenza virus, respiratory syncytial virus, mumps virus and measles virus), Rhabdoviridae (including, for example, rabies virus), Bunyaviridae (including, for example, hantavirus), Orthomyxoviridae (including, for example, influenza A, B and C), Retroviridae (including, for example, HIV and HTLV), and Hepadnaviridae (including, for example, hepatitis B virus). In a more preferred example, the antigen is from a pathogen causing disease in livestock and poultry, especially from viral pathogens including, for example, reovirus (e.g., african horse sickness virus or bluetongue virus) and herpes virus (including equine herpes virus). The antigen may be an antigen from foot and mouth disease virus. In a more preferred embodiment, the antigen is from tick-borne encephalitis virus, dengue virus, SARS, West Nile virus and Hantaan virus.

In another preferred embodiment, the antigen is from the family of Retroviraae (e.g., HTLV-I, HTLV-11 or HIV-1 (also known as HTLV-111, LAV, ARV, hTLR, etc.)). Especially from HIV, especially the isolates HIVI11b, HIVSF2, HTVLAV, HIVLAI, HIVMN; HIV-1CM235, HIV-1; or HIV-2. In a particularly preferred embodiment, the antigen is a Human Immunodeficiency Virus (HIV) antigen. Examples of preferred HIV antigens include, for example, gp120, gp 160 gp41, gag antigens such as p24gag and p55gag, and proteins derived from pol, env, tat, vif, rev, nef, vpr, vpu, or LTR regions of HIV. In a particularly preferred embodiment, the antigen is HIV gp120 or a portion of HIV gp 120. The antigen may be from an immunodeficiency virus, for example from SIV or feline immunodeficiency virus.

Thus, the encoded polypeptide may be an antigen, an immunogenic fragment thereof, or an immunogenic variant thereof. For example, the fragment or variant may have any level of homology, any proportion of the length of the original antigen, and the functions described herein, particularly the ability to generate an immune response. In some cases, the coding sequence of the nucleic acid construct may be modified to optimize expression. For example, the codon usage can be modified to that typical for the subject. The subject's Kozak consensus sequence may also be replaced by a naturally occurring sequence surrounding the start codon.

In a particularly preferred embodiment, the nucleic acid construct of the invention comprises a coding sequence encoding an immunogenic variant of an influenza antigen, an immunogenic fragment thereof, or both. Fragments and/or variants can have any level of sequence homology, fragment length, and/or functional level described herein. In particular, the coding sequence of the preferred construct encodes an influenza virus antigen, an immunogenic fragment of an influenza virus antigen, or an immunogenic variant having 80% amino acid homology to both.

For example, the influenza antigen may be influenza NP (nucleoprotein/nucleocapsid protein), HA (hemagglutinin), NA (neuraminidase), M1, M2, PB1, PB2, PA, NS1 and/or NS2 antigens, or may be fragments or variants of these antigens. In a preferred embodiment, the encoded antigen may be an HA, NA and/or M2 influenza antigen or a fragment or variant of such an antigen. In a particularly preferred example, the encoded antigen may be an HA or NA antigen or a fragment or variant of these antigens, in particular an HA antigen or a fragment or variant of this antigen.

Thus, in a particularly preferred construct of the invention, the encoded antigen may be influenza Hemagglutinin (HA), an immunogenic fragment thereof, or an immunogenic fragment having 80% amino acid sequence homology thereto. In a more preferred example, the encoded antigen is influenza neuraminidase, M2, an immunogenic fragment of both, or an immunogenic fragment with 80% amino acid sequence homology thereto.

In a preferred example, a construct of the invention may encode more than one polypeptide, in particular more than one influenza antigen, an immunogenic fragment thereof, or an immunogenic variant of both. In the case where more than one antigen is applied to the preferred examples, HA and NA antigens or fragments or variants of these antigens may be used together.

Where the constructs of the invention express more than one influenza antigen, immunogenic fragment thereof, or immunogenic variant of both, at least two different encoded antigens, fragments or variants are from the same influenza polypeptide of different influenza strains.

In a preferred embodiment, the antigen may be from the H5N1 strain of influenza virus, and immunogenic fragments thereof or immunogenic-retaining variants of both may also be used. In particular, the antigen may be an antigen from the H5N1 strain or a fragment of the antigen.

In certain instances, the antigen may be a fragment or variant of a naturally occurring influenza virus polypeptide. For example, the antigen may correspond to a subregion of a naturally occurring influenza polypeptide, including any of the different fragment lengths described herein. The antigen may be a variant of a naturally occurring influenza virus antigen or a fragment of the antigen. Preferably, the variant and/or fragment is capable of generating an immune response which recognises the antigen, particularly the influenza virus antigen from which the fragment or variant is derived.

The influenza antigen may be from any influenza virus. The antigen may be from an influenza a, b or c virus, in particular from an influenza a and/or b virus. The antigen may be from a variant influenza virus strain, in particular a variant strain associated with increased infectivity or pathogenicity of an influenza virus strain. For example, the antigen may be from one of the strains identified annually by the world health organization for influenza virus vaccines, in particular, the antigen may be one identified by the WHO for such use. In a preferred example, a nucleic acid construct, population of nucleic acid constructs, pharmaceutical composition or vaccine of the invention may encode antigens of three influenza virus strains, in particular each of the three influenza strains identified by WHO or other comparable authorities over a particular year.

In a preferred example, the one or more encoded influenza antigens may be from an influenza virus strain. Thus, the immunogenic variant of an influenza antigen, an immunogenic fragment thereof, or both, can be from an influenza strain. For example, a construct encoding an antigen from an epidemic strain may be administered or present alone. In other cases, the construct may also encode or be administered with other constructs that encode other antigens. In a preferred example, the antigen from an influenza strain and the antigen from a non-influenza strain may be encoded in the same or different constructs. Specifically, an influenza antigen and an antigen from 1, 2, 3, 4, 5, 6 or more non-influenza virus strains may be administered, preferably an antigen from each of 3, 4 or 5 non-influenza virus strains, more preferably an antigen from each of 3 or 4 non-influenza virus strains.

In certain instances, a construct may encode more than one polypeptide. In particular, the construct may encode more than one antigen, in particular more than one influenza antigen. For example, the construct may express two, three, four, five, six or more polypeptides, particularly antigens. In a preferred example, the construct may encode three, four, five or more different polypeptides, in particular antigens. In a more preferred embodiment, the construct may encode three, four or five different polypeptides, in particular antigens. In a more preferred embodiment, the construct may encode three or four different polypeptides, in particular antigens, in particular influenza antigens. At least one antigen, and preferably all antigens, can be expressed using the chimeric promoter of the present invention. Typically, each antigen is expressed from a different chimeric promoter of the invention. In some cases, one promoter may be used to form transcripts that produce multiple polypeptides. In some cases, several antigens may be expressed as one fusion protein.

In a preferred example, the constructs of the invention encode an immunogenic variant of an influenza antigen, an immunogenic fragment thereof, or both, and one or more non-influenza antigens, immunogenic fragments thereof, or immunogenic variants of both.

The nucleic acid constructs of the invention are useful in vaccines. Accordingly, the present invention provides a vaccine composition comprising a nucleic acid construct, a population of nucleic acid constructs or coated vector particles of the invention. The vaccine may comprise a suitable pharmaceutical carrier or excipient. The vaccine may comprise an adjuvant, in particular an adjuvant construct of the invention. Alternatively, the vaccine may be administered separately, sequentially or simultaneously with this adjuvant.

In a preferred embodiment, the present invention provides a multivalent vaccine comprising at least two different constructs of the invention encoding different antigens, immunogenic fragments thereof, or immunogenic variants of both. The antigen can be any antigen described herein. In a preferred embodiment, the present invention provides a multivalent vaccine comprising at least two different constructs of the invention encoding different influenza antigens, immunogenic fragments thereof, or immunogenic variants of both. In other cases, multiple antigens may be expressed from the same construct to provide a multivalent vaccine, or a combination of constructs encoding one or more antigens may be used.

In a more preferred example, the multivalent vaccine of the invention may be a multivalent vaccine of a trivalent, tetravalent or pentavalent vaccine encoding three, four or five different antigens, immunogenic fragments or immunogenic variants, in particular variants of influenza antigens, fragments thereof or both.

In another preferred example, vaccines of the invention, including multivalent vaccines, may include constructs that encode immunogenic variants of influenza antigens, immunogenic fragments thereof, or both. A multivalent vaccine may encode an immunogenic variant of an influenza antigen, an immunogenic fragment, or both, and may also encode, for example, an antigen from each of three, four, or five different influenza virus strains, an immunogenic fragment thereof, or immunogenic variants of both.

Adjuvants may be present in the vaccine of the invention or administered simultaneously, separately or sequentially with the vaccine of the invention. In particular, the invention provides a vaccine comprising at least one construct of the invention encoding an ADP ribosylated bacterial toxin subunit, a fragment thereof having adjuvant activity or a variant thereof having adjuvant activity.

Nucleic acid constructs expressing more than one antigen may be used to form multivalent vaccines, i.e. vaccines intended to immunize against a variety of different antigens, in particular against influenza antigens. In some cases, one or more influenza antigens may be provided, as well as one or more antigens from different pathogens. Thus, any of the constructs, populations of nucleic acid constructs, vaccines, pharmaceutical compositions, and coated vector particles described herein can comprise a construct encoding an influenza antigen, and the same construct or different constructs can encode antigens from different pathogens. The various multivalent influenza constructs, populations, and vaccines described herein may also encode one or more antigens from different pathogens.

In some cases, the encoded antigens are from the same pathogen. In some cases, the different antigens are all from the same virus. Thus, in a preferred embodiment, all antigens are influenza antigens. The multiple antigens may be from the same strain of influenza virus and thus may be from several different polypeptides of influenza virus. Alternatively, the multiple antigens may be from different strains of virus, particularly influenza virus. For multivalent constructs and/or vaccines, antigens from at least two, preferably at least three, four or five different influenza virus strains may be used. For example, the antigen may be from 2, 3, 4, 5, 6 or more different strains, in particular antigens from three, four or five strains may be used, more preferably from three or four different strains.

Alternatively or additionally, a multivalent vaccine may comprise a plurality of different constructs of the invention, the different constructs encoding different antigens. For example, at least two, three, four, five or six different constructs may be used. In a particularly preferred embodiment, two, three, four or five different constructs, in particular three or four constructs, can be used. In one example, if more than one construct is present, each construct encodes only one antigen. In another example, the construct may encode two, three, four, five or more different antigens, in particular three or four different antigens.

The invention also provides a population of nucleic acid constructs, wherein the population comprises a plurality of constructs of the invention, particularly any combination of the foregoing. Thus, the invention provides a population of nucleic acid constructs, wherein the population comprises at least two different constructs of the invention. The nucleic acid constructs are present in any suitable amount relative to each other. Typically, each nucleic acid construct is present in a weight ratio of about 1: 10 relative to each other.

The population of nucleic acid constructs can be used to form a multivalent vaccine and can be used in various methods of the invention. Multivalent vaccines are multivalent in that they may also include any construct of the invention that encodes more than one antigen.

In a preferred embodiment, a population of nucleic acids is provided, the population comprising at least two different constructs encoding different antigens. In particular, the invention provides a population comprising at least two different constructs encoding an influenza virus antigen, an immunogenic fragment thereof, or immunogenic variants of both. In this population of nucleic acid constructs, it is preferred that the at least two different antigens are from the same influenza polypeptide, e.g., HA, of different influenza virus strains. In populations where at least two different constructs encoding influenza antigens are present, it is preferred that the at least two different antigens, fragments or variants are from different influenza polypeptides derived from the same or different influenza virus strains. Another preferred population comprises a population of nucleic acid constructs, wherein the population comprises at least three different constructs, each of which encodes an antigen or immunogenic fragment or immunogenic variant of a different influenza virus strain.

In another preferred embodiment, the invention provides a population of nucleic acid constructs comprising at least one construct encoding an immunogenic variant of an antigen, an immunogenic fragment thereof, or both; and at least one construct encoding an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof having adjuvant activity. The or each such construct encoding an antigen, immunogenic fragment or immunogenic variant is typically present in a weight ratio of about 10: 1 to 1: 10 relative to the or each such construct encoding an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity or a variant thereof having adjuvant activity. The weight ratio can be from about 10: 1 to about 1: 1, for example about 9: 1.

In cases where multiple polypeptides are encoded, any combination of constructs encoding one or more polypeptides and multiple constructs may be used. For example, where three antigens are encoded, one construct may encode all three antigens, one construct encodes two antigens and the other construct encodes one antigen, or three constructs, each encoding one antigen, may be used, for example. Where four antigens are encoded, they may be encoded by four, three, two or one construct, each construct encoding one, two, three or four different antigens. In one example, as few constructs as possible are used, e.g., only one or two constructs are used. The invention also provides vaccines and populations of nucleic acid constructs comprising these combinations of nucleic acid constructs.

In a preferred embodiment, the invention provides a multivalent vaccine or population of nucleic acids comprising a construct encoding an antigen of an influenza virus and encoding 3, 4 or 5, especially 3 or 4, non-influenza antigens. The non-influenza antigen may be encoded by the same or different construct as that encoding the influenza antigen. In a preferred example, the 3, 4 or 5 other non-influenza antigens may be encoded by one or more different constructs, in particular by only one different construct. In another example, a construct encoding an epidemic antigen may also encode one or more additional antigens.

In some embodiments, the nucleic acid construct may encode an adjuvant, or be encoded by a different construct. Any of the populations, compositions and vaccines of the invention may include such adjuvant constructs or be administered simultaneously, sequentially or separately. Thus, a sequence that has been inserted into a cloning site for insertion of a coding sequence may encode a polypeptide that acts as an adjuvant.

Thus, in one example, the invention includes a nucleic acid construct wherein the coding sequence encodes a variant of an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or both, having adjuvant activity and also having 80% amino acid homology thereto. In a preferred example, the nucleic acid construct comprises two coding sequences comprising an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant of both having adjuvant activity, wherein each coding sequence is linked to such a chimeric promoter.

In one example, the two coding sequences encoding the ADP-ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant of both having adjuvant activity may be in opposite directions. In another example, two coding sequences encoding ADP-ribosylating bacterial toxin subunits, fragments thereof having adjuvant activity, or variants of both having adjuvant activity may be in the same orientation. In any construct of the invention comprising more than one coding sequence linked to a chimeric promoter, the promoters may be in the same orientation or in different orientations, or, when three or more such promoters are present, may be some in the same orientation and some in different orientations.

In a preferred example, the adjuvant encoded in either adjuvant construct may be an ADP ribosylating bacterial toxin. These include Diphtheria Toxin (DT), Pertussis Toxin (PT), Cholera Toxin (CT), escherichia coli heat labile toxin (e.g. c. heat toxin, LT1 and LT2), pseudomonas endotoxin a (pseudomonas exotoxin a), pseudomonas exotoxin s (pseudomonas exotoxin s), cactus extracellular enzyme (B. cereus exotzyme), bacillus sphaericus toxin (B. sphaericus toxin), clostridium botulinum C2 and C3toxin (C botulinum C2 and C3toxin), clostridium mucronatum extracellular enzyme (C. limosum exotzyme), and toxins from clostridium perfringens (C. perfringens), clostridium spirochetum (C. spirofa) and clostridium difficile (C difficile) and staphylococcus aureus staphylococcus n staphylococcus aureus toxin. Most ADP-ribosylated bacterial toxins contain an a subunit and a B subunit. The construct may express the a subunit, the B subunit, and/or both subunits together.

In a preferred embodiment, the nucleic acid construct may encode an E.coli heat-labile toxin and/or a cholera toxin, particularly for expression of an E.coli heat-labile toxin. The GenBank entry for the complete sequence of the a and B subunit genes of CT is found at locus vibcrxabb (accession No. d30053), while the GenBank entry for the complete sequence of the a and B subunit genes of LT is found at locus AB0116677 (accession No. ab011677). In a particularly preferred example, the construct of the invention may encode the a and/or B subunit of LT encoded by the vectors pPJV2012 and/or pjv7788, or a fragment of this subunit that retains adjuvant activity, or a variant of both that retains adjuvant activity. Constructs of the invention may comprise coding sequences for the LTA subunit and/or LTB subunit of vectors pPJV2012 and/or pjv7788, fragments thereof encoding polypeptides having adjuvant activity, or variants of both having adjuvant activity. In a preferred embodiment, the construct of the invention may have both an LT a coding sequence and an LTB coding sequence.

In another preferred example, the construct may comprise a nucleic acid sequence consisting of SEQ ID No: 61 or a sequence having 60% sequence identity thereto. In another example, the construct may comprise a nucleic acid sequence consisting of SEQ ID No: 62 or a sequence having 60% sequence identity thereto.

The construct may express active variants or fragments of a particular adjuvant. A variant or fragment is active if it retains at least a portion of the adjuvant activity of the polypeptide from which it is derived. Thus, the variants and/or fragments can still enhance the immune response to a particular antigen compared to that observed when the antigen is administered without an adjuvant. The encoded sequence may be an active fragment or variant of the a and/or B subunit of CT, in particular an active fragment of the a and/or B subunit of LT. For example, variants and fragments that can be used can have any length of the variants and fragments, any level of sequence homology, or other features described herein. For example, they can be evaluated by differential immunogenicity analysis using specific antigens and then comparing the observed results with adjuvant base and variant vectors encoding adjuvants. In a particularly preferred embodiment, the construct may encode the LTA and/or LTB subunit, in particular both. In a particularly preferred example, the construct may be a pPJV2012 or pjv7788 of the sequences provided herein.

Any of the adjuvant constructs of the invention may be administered simultaneously, sequentially or separately with the antigen or nucleic acid encoding the antigen. In a preferred embodiment, the adjuvant construct of the invention may be administered simultaneously, sequentially or separately with the construct of the invention encoding the antigen. Compositions and vaccines containing the adjuvant construct and the construct encoding the antigen may be provided in the form of a core vector coated with both types of constructs provided on its surface or in the form of a mixture of a core vector coated with the adjuvant construct and another core vector coated with the construct encoding the antigen.

The toxin subunit may lack the naturally occurring signal sequence. The native signal sequence of the exotoxin may be replaced by a signal sequence of a eukaryote, particularly the chicken lysozyme signal peptide. Naturally occurring exotoxin subunits can be modified to detoxify the toxin. The a subunit can be modified to interfere with the activity of or inactivate an ADP-ribosyltransferase. In some cases, the exotoxin subunits remain toxic. Thus, in some cases, the exotoxin subunits may not be detoxified.

Thus, the adjuvant constructs of the invention may be used to enhance an immune response against a particular antigen. An enhanced immune response includes a more intense or longer immune response. This means that when the antigen is encountered again, the immune response is stronger than when no adjuvant is administered. An enhanced immune response may result in higher antibody titers. For certain constructs, particularly those expressing exotoxin subunits, adjuvants can generate amplified cellular responses to the antigen of interest as well as T helper 1-like immune responses.

The adjuvant construct may be administered with any of the other constructs described herein, present in a vaccine with the other constructs, or present in a population of nucleic acids with the other constructs. The adjuvant construct may also encode or be administered with a construct encoding one or more antigens including any of those described herein, particularly antigens encoding influenza virus.

In one embodiment, the construct of the invention may encode an immunostimulant and/or an immunosuppressant. In a preferred example, the construct may encode one or more of the following: alpha-interferon, beta-interferon and/or gamma-interferon, interleukin-1, interleukin-2, interleukin-4, interleukin-5, interleukin-7, interleukin-10, interleukin-12, interleukin-13, interleukin-18, interleukin-23 and interleukin 24, GM-CSF, G-CSF, TGF-beta, B7.1, B7.2, CTLA-4, CD40 ligand, CD40, OX40, OX40 ligand, Flt-3 ligand, TRAIL, TRANCE, Fas ligand, TNF-alpha, MCP-1 alpha, PF-4, SLC, MIP-3 alpha, IP-10. In certain cases, such a construct may be administered simultaneously, separately or sequentially with other constructs, in particular one of the constructs of the invention, in particular the construct encoding the antigen.

The constructs of the invention may comprise a polyadenylation signal. The nucleic acid inserted into the cloning site may include a polyadenylation (polyA) sequence. Such PolyA sequences are typically derived from coding sequences. Alternatively, a heterologous polyA sequence may be provided in a nucleic acid construct of the invention. Typically, the polyA sequence will be located downstream of the cloning site so that it can be operably linked to the coding sequence inserted into the cloning site. Any suitable polyA sequence may be introduced into the construct using standard cloning techniques. Such polyA sequences are well known in the art.

The poly A sequence may be:

(i) a native polyA sequence;

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

The native polyA sequence (i) may be, for example, the rabbit β -globin gene polyA, the Human Papilloma Virus (HPV) early or late gene polyA, the HSV-2gB gene polyA, the simian CMV immediate early gene polyA or the HSVgD late gene polyA. The polyadenylation sequence may be derived from the polyadenylation sequence of a gene selected from: rabbit β -globin gene, Human Papilloma Virus (HPV) early or late gene, HSV-2gB gene, simian CMV immediate early gene, and HSVgD late gene.

Preferably, the native polyA sequence (i) is selected from SEQ ID No.10(GenBank K03256), SEQ ID No.11(GenBank M16019), SEQ ID No.12(GenBank Z80699) and SEQ ID No.13(GenBank K02718). Particularly preferred polyA sequences include SEQ ID NOs: 54, or a functionally homologous variant or fragment thereof. Preferred poly A sequences are also provided by nucleotides 2556 to 2686 of SEQ ID No.14, or functional fragments thereof or functional variants of both may also be used.

In a more preferred example, the polyadenylation signal used in the construct of the invention may comprise:

(i) nucleotide sequence SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID No: 54 from nucleotide 4243 to 4373 of SEQ ID No: nucleotides 906 to 1038 of 61, SEQ ID No: 4375 to 4050 nucleotides of 61 and/or SEQ ID No: nucleotides 2463 to 2593 of 62;

(iii) (iii) a functional fragment of (i) or (ii).

Generally, a functional polyA sequence is a sequence that retains polyadenylation activity.

The ability of polyA sequences to polyadenylate RNA transcripts can be tested using conventional expression assays. In the detection, it is found that: the functionally homologous variant (ii) or functional fragment (iii) typically has at least 50%, e.g. 60%, 70%, 80% or more of the polyA activity of the native polyA sequence.

In general, expression will be enhanced when a polyA sequence is present in a construct of the invention, typically resulting in an enhancement comparable to the native polyA sequence (i) described above.

The functionality of polyA sequences can be detected using the differential expression assay described below. The polyA region to be tested is introduced into the basic vector to replace RBGpA. In contrast to the base vector, a polyA to be tested is considered functional if the polyA does not stop expression (preferably increases expression) in at least one (preferably both) of the reference cell types. Preferably, expression is increased by at least 5%, 10%, 20%, 30%, 40% or 50% or more. The degree of enhancement may be any of the levels described herein. Reduction may occur in certain circumstances, including any of the levels mentioned herein. Preferred cell types are mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cells. SSC15 or B16 cells can also be used. According to the present assay, a homologous variant (ii) or fragment (iii) is generally considered functional if it can achieve an increase in expression of more than 50% of that obtained for the native polyA sequence (i).

Alternatively or additionally, the activity of the polyA sequence may be detected using the following differential immunogenicity assay. The polyA sequence was introduced into the basic vector to replace RBCpA. Functional polyA sequences provide antibody titers that are as high or higher than those obtained with the basic vector having at least one, and preferably two, antigens. Preferably, the antibody titer is at least 5%, 10%, such as 20%, 30% or 40% higher than the antibody titer obtained with the base vector. In some cases, any increase or decrease described herein can be observed. Preferred antigens are HBsAg, HSV2gD and Flu-M2 antigen. Particularly preferred antigens are influenza virus antigens, including any of the influenza virus antigens mentioned herein, in particular the HA, NA and M2 antigens. In particular, an HA antigen, or an immunogenic fragment thereof, or an immunogenic variant of both, may be used. The homologous variant (ii) or fragment (iii) is generally functional if it can achieve the highest antibody titre achieved by the native polyA sequence (i).

The nucleic acid construct may comprise additional regulatory sequences which affect the expression of the coding sequence inserted into the cloning site. The construct may include an untranslated leader sequence. This sequence is operably linked to the chimeric promoter in the construct, and thus it is also operably linked to the coding sequence inserted into the cloning site. The leader sequence provides a translation initiation site for expression of the inserted coding sequence and typically contains a Kozak sequence.

Generally, the untranslated leader sequence includes:

(i) a native non-translated leader sequence;

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

Typically the leader sequence will comprise a sequence present in (i) which binds to the transcription component or regulatory protein, or a homologue of such sequences which is capable of binding to the same component or protein. Typically the sequence or homologue thereof is present in the leader sequence in the same order and/or at substantially the same relative spacing as in (i). Typically, the leader sequence contains a translation initiation site for expression of the inserted coding sequence. Typically, the leader sequence contains a Kozak sequence.

Generally, the untranslated leader sequence is about 10 to about 200 nucleotides in length, e.g., about 15 to 150 nucleotides, preferably 15 to about 130 nucleotides. For example, leader sequences containing 15, 50, 75 or 100 nucleotides may be used.

Typically, a functional untranslated leader sequence is a leader sequence that provides a translational start site for expression of a coding sequence operably linked to the leader sequence. Typically, the functional variant (ii) or fragment (iii) has substantially the same activity as and/or complements the activity of the native sequence (i), which activity typically contributes to or enhances expression of a coding sequence to which the sequence is operably linked.

The activity of variant (ii) or fragment (iii) as an untranslated leader sequence relative to the native leader sequence can be tested using standard methods. For example, expression vectors containing the native leader sequence (i) operably linked to its native coding sequence may be prepared and expression monitored in a suitable host cell, e.g., a mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cell, etc. Test constructs in which the native leader sequence is replaced by homologous variants or fragments can be prepared and expression monitored in the same host cell. Typically, the variant (ii) or fragment (iii) may provide at least 50%, for example 60%, 70%, 80%, 90% or 100% or more of the expression provided by the native sequence. In certain instances, an increase or decrease in any of the expressions described herein can be observed.

The utility of possible leader sequences can also be tested in the following differential expression analysis. The test leader sequence was introduced into the basic vector to replace the HBV pre S25' UTR. The functional leader sequence does not stop expression, but preferably increases expression, in at least one (preferably both) reference cell types compared to the base vector. Expression is typically increased by at least 5%, 10%, 20%, 30%, 40% or 50%. Preferred cell types are mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cells. According to the present analysis, a homologous variant (ii) or fragment (iii) is generally considered functional if it can obtain more than 50% of the expression increase obtained by the native leader sequence.

Alternatively or additionally, the leader sequence may be tested for activity in the differential immunogenicity assay described below. Leader sequences were introduced into the basic vector to replace the HBV pre S25' UTR. The functional leader sequence provides an antibody titer which is at least as high as or higher than the antibody titer obtained with the base vector having at least one (preferably two) antigens. Preferably, the antibody titer is at least 5%, 10%, 20%, 30% or 40% higher than the antibody titer of the base vector. Preferred antigens are the HBsAg, HSV 2gD and Flu-M2 antigens. Particularly preferred antigens are influenza virus antigens, including any of the influenza virus antigens mentioned herein, especially the HA, NA and M2 antigens. In particular, HA antigens or fragments or variants thereof may be used. The homologous variant (ii) or fragment (iii) is generally functional if it can achieve the highest antibody titre achieved by the native leader sequence (i). However, in some cases, expression of any of the reduced levels described herein may be observed.

Suitable leader sequences may be obtained using standard methods. For example, HBV preS2 antigen sequence, HBV e-antigen sequence, and HSV-type 2gD antigen sequence are available in GenBank M54923, and Z86099, respectively. Primers can be designed based on this known sequence and used to isolate homologous sequences. The leader sequence can be synthesized based on known sequences.

In general, the native sequence (i) is a eukaryotic sequence or a viral sequence, in particular a virus that infects eukaryotes. Preferably, native sequence (i) is an HBV or HSV sequence, such as an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence. Particularly preferably, (i) is selected from the group consisting of SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7. In a preferred example, the untranslated leader sequence comprises SEQ ID NO: 54 or 1864-1984 nucleotides of SEQ ID No. 14. Functional fragments or variants of any of these sequences may also be used.

In one example, the untranslated leader sequence includes:

(i) nucleotide sequences SEQ ID No 5, SEQ ID No: 6. seq ID No: 7, or nucleotides 1864 to 1984 of seq id No. 54;

(ii) (ii) a functional variant of (i) which has at least 80% nucleotide sequence homology with (i); and/or

(iii) (iii) a functional fragment of (i) or (ii).

The untranslated leader sequences can be used in any construct of the present invention.

The nucleic acid construct may include an enhancer sequence. Enhancer sequences are typically provided 3' to the cloning site, operably linked to the chimeric promoter and the inserted coding sequence, and serve to increase transcription of the inserted sequence.

Typically, enhancers include:

(i) a natural enhancer;

(ii) (ii) a functionally homologous variant of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

Enhancer sequences typically contain from about 50 to about 850 nucleotides, for example from about 75 to about 500 nucleotides. Enhancers of about 100, 200, 300, or 400 nucleotides may be used.

In general, (i) is an enhancer of a eukaryote or a virus, particularly a virus that infects a eukaryote. Usually these enhancers are present in the 3 'untranslated region (3' UTR) of a gene. Preferably (i) is an HBV or CMV enhancer, such as HBs Ag3 'UTR or simian CMV immediate early gene 3' UTR. Preferably (i) comprises SEQ ID No.8 or SEQ ID No. 9. Preferably (i) comprises SEQ ID NO: 54 nucleotide 3699-4231. In another preferred example, (i) may comprise SEQ ID NO: nucleotide 2012 to 2544 of 14. Functional fragments or variants of any of these sequences may be used.

Typically, the enhancer in the construct comprises sequences present in (i) that bind to a transcription component or regulatory protein, such as a transcription factor, or homologues of such sequences that bind to the same component or protein. Preferably, these sequences are present in the enhancer in the same order and/or substantially the same relative spacing as in (i).

Generally, a functional enhancer is an enhancer that enhances or increases the expression of a polynucleotide, such as a coding sequence or the like, to which the polynucleotide is operably linked. Typically, the functional homologous variant (ii) or fragment (iii) has substantially the same activity (e.g. enhances expression) as and/or complements the activity of the native enhancer (i).

Enhancer activity can be assayed using an enhancer trap assay (enhancer trap assay). Protocols are known in the art. In this assay, the functionally homologous variant (ii) or fragment (iii) preferably provides at least 50% of the enhancer activity shown by the native enhancer. Typically the activity is at least 60%, 70%, 80%, 90, 100% or more of the activity of the native enhancer. Generally, in this assay, the functional variant (ii) or fragment (iii) complements the activity of the native enhancer (i). For example, any increase or decrease described herein can be observed.

In a preferred embodiment, the enhancer sequence comprises:

(i) nucleotide sequence SEQ ID No: 8. SEQ ID No: 9, nucleotides 3699 to 4231 of SEQ ID No.54, SEQ ID No: nucleotides 3831 to 4363 of 61 and/or SEQ ID No: nucleotides 4507 to 5038 of 62;

(ii) (ii) a functionally homologous variant of (i) having at least 80% nucleotide sequence homology with one or more sequences of (i); and/or

(iii) (iii) a functional fragment of (i) or (ii).

The utility of enhancers can also be tested using the differential expression analysis described below. Test 3' UTR sequences were introduced into the basic vector. In this assay, it is of utility if the expression of the 3' UTR is not halted, preferably increased, in at least one, preferably two, reference cell types as compared to the base vector. Preferably expression is increased by at least 5%, 10%, 20%, 30%, 40% or 50%. Preferred cell types are mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cells. According to this analysis, a homologous variant (ii) or fragment (iii) is functional if it can obtain more than 50% of the expression increase obtained by the native enhancer sequence (i).

Additionally or alternatively, the activity of the enhancer sequence may be detected in the differential immunogenicity assay described below. The 3' UTR was introduced into the basic vector. The functional enhancer sequence provides antibody titers that are as high or higher than those provided by the basic vector with at least one (preferably two) antigens. Preferably, the antibody titer is increased by at least 5%, 10%, 20%, 30% or 40% over that provided by the base vector. Preferred antigens are HBs Ag, HSV2gD and Flu-M2 antigen. Particularly preferred antibodies are influenza virus antigens, including any of the influenza virus antigens described herein, particularly the HA, NA and M2 antigens. In particular, HA or fragments or variants thereof may be used. A homologous variant (ii) or fragment (iii) is functional if it has the highest antibody titer of the native enhancer sequence (i).

Suitable enhancer sequences can be obtained using standard cloning methods. For example, HBs Ag 3 'UTR sequences or simian CMV immediate early gene 3' UTR sequences are available in genbank af143308 and M16019. Primers can be designed based on known sequences and used to isolate homologous sequences.

In some cases, if multiple polypeptides are encoded by a construct, it may be desirable to delete specific sequences to reduce the size of the construct. In particular, the construct may lack an untranslated leader coding region and/or enhancer operably linked to one or more coding sequences for the polypeptide to be expressed. For example, this may be the case when three, four, five or more peptides are expressed from the same construct, in particular three, four or five polypeptides.

In a preferred embodiment, the nucleic acid construct comprises a heterologous polyA sequence, a heterologous leader sequence, and a heterologous enhancer, all operably linked to a chimeric promoter for efficient expression of an inserted coding sequence.

In a further aspect, the invention also provides a nucleic acid construct comprising or consisting essentially of:

(i) a promoter sequence;

(iii) (iii) a coding sequence operably linked to (i) and (ii)

Typically, the promoter sequence (i) is derived from a viral or eukaryotic promoter sequence. The promoter sequence may be a native promoter sequence, a functional homologue of a native sequence, or a functional fragment of both. Suitable natural promoters include, for example, the hCMV immediate early promoter, the pseudorabies virus (PRV) promoter, or the Rous Sarcoma Virus (RSV) promoter. Preferably, the native promoter comprises SEQ ID NO: 52 or SEQ ID NO: 53.

an artificial promoter construct such as the chimeric promoter described above may be used as long as the promoter is functional.

A functional promoter sequence is generally a promoter sequence that is capable of causing (including promoting and controlling) transcription of an operably linked coding sequence in an appropriate host cell.

Promoter activity of the promoter sequence can be detected using conventional expression assays. In this assay, a functional homologue or fragment of the native promoter sequence typically provides at least 50%, e.g., at least 60%, 70%, 80%, or 90% of the expression provided by the native sequence.

The untranslated leader sequence (ii) is as described above. Suitable coding sequences (iii) include those which are described in relation to the parts of the chimeric promoter construct. However, in this aspect of the invention, the coding sequence is heterologous to the untranslated leader sequence. The constructs of the invention typically comprise a polyA sequence, which, as noted above, may be derived from a coding sequence, or provided in the construct as a heterologous polyA sequence. Suitable polyA sequences have been described. The construct also includes an enhancer sequence located 3' to the coding sequence. Suitable enhancer sequences have been described above in relation to the chimeric promoter construct.

In another aspect, the invention provides a nucleic acid construct comprising or, in certain embodiments, consisting essentially of:

(i) a promoter sequence;

(ii) (ii) a coding sequence operably linked to the promoter sequence (i); and

wherein the enhancer sequence (iii) is derived from the 3 'UTR of the HBsAg sequence or the 3' UTR of the simian CMV immediate early gene sequence and the coding sequence (ii) is heterologous to the enhancer sequence.

The construct may include untranslated leader sequences such as those already described in chimeric promoter constructs.

Typically, the promoter sequence (i) is derived from a viral or eukaryotic promoter sequence. The promoter sequence may be a native promoter sequence, a functional homologue of a native sequence, or a functional fragment of both. For example, suitable natural promoters include the hCMV immediate early promoter, the pseudorabies virus (PRV) promoter, or the Rous Sarcoma Virus (RSV) promoter. Preferably, the native promoter comprises SEQ ID NO: 52 or SEQ ID NO: 53.

A functional promoter sequence is generally a promoter sequence that is capable of causing (including promoting and regulating) transcription of an operably linked coding sequence in a suitable host cell.

Suitable coding sequences (ii) include those already mentioned in relation to the parts of the chimeric promoter construct. However, in this aspect, the coding sequence is heterologous to the 3' enhancer sequence. The enhancer sequence (iii) of this construct is as described above. The constructs of the invention also typically include a polyA sequence. As in the chimeric promoter construct, the polyA region may be derived from coding sequence (ii) or may be a heterologous polyA component in the construct.

The construct according to any aspect of the invention may comprise a signal peptide sequence. Thus, a nucleic acid construct may comprise a nucleotide sequence encoding a signal peptide operably linked to the coding sequence. The signal peptide sequence is inserted in operative linkage with a promoter, whereby the signal peptide is expressed and secretion of the polypeptide encoded by the coding sequence also in operative linkage with the promoter is facilitated.

Typically, the signal peptide sequence encodes a peptide of 10 to 30 amino acids, such as 15 to 20 amino acids. Typically amino acids are predominantly hydrophobic. Typically, the signal peptide directs the synthesizing polypeptide chain with the signal peptide into the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved in the endoplasmic reticulum, and the polypeptide is secreted through the Golgi apparatus.

Signal peptides for use in the present invention may include:

(i) a native signal peptide sequence;

(ii) (ii) a homologous variant of (i) which retains signal peptide activity; or

(iii) (iii) a fragment of (i) or (ii) which retains signal peptide activity.

For example, sequence (i) may be human tissue plasminogen activator signal peptide (hTPASP) (GenBank L00141), aprotinin signal peptide (GenBank AAD13685), tobacco extensin signal peptide (GenBank JU0465), or chicken lysozyme signal peptide (GenBank AF 410481).

The signal peptide suitable for use in the present invention is a signal peptide capable of secreting a heterologous protein. Functional signal peptides can be identified in assays that compare the effect of a signal peptide to be tested to the effect of a known signal peptide, such as human tissue plasminogen activator signal peptide (hTPPasp), to the effect of no signal peptide. The differential expression analysis described below can be used, but requires the following modifications. Constructing a secretion expression vector with a signal peptide to be detected, hTPPasp or without the signal peptide in a basic vector. The coding sequence for the polypeptide lacking its naturally occurring signal peptide is inserted into a vector, and the vector is transformed into a reference host cell. Preferred cells are mammalian HEK293T, CHO, HeLa, BHK, 3T3 or COS cells. Analyzing the cell culture medium to obtain the expression level of the polypeptide. Functional signal peptides enable secretion of polypeptides at higher levels compared to vectors with at least one, preferably two polypeptides lacking the signal peptide. Typically, secretion is increased by 5%, or more preferably by 10% or more, such as by 20% or 50% or more. Generally, the secretion level is comparable to that obtained using hTPAsp. In some cases, any increase or decrease described herein can be observed.

In a preferred embodiment, the signal peptide is selected from the group consisting of human tissue plasminogen activator signal peptide (hTPASP), aprotinin signal peptide, tobacco extensin signal peptide, and chicken lysozyme signal peptide.

The encoded protein can be secreted extracellularly for a number of advantages, particularly when the protein is an antigen. For example, increased secretion of antigens provides the possibility for more antigen uptake and response by immune cells (macrophages, langerhan cells, B cells, T cells, etc.), giving antigens the ability to reach the blood stream and signal cells (cytokines), enabling antigens to find cellular ligands and function (antibodies, toxins such as cholera toxin, e.g. e.coli LT), and participating in normal cellular biochemical processes (cellular receptors).

The nucleic acid construct of the present invention may be in the form of a plasmid. The nucleic acid construct of the present invention may be in the form of a plasmid expression vector. In a preferred embodiment, the construct of the invention may be a DNA construct. In other cases, the construct may be an RNA or PNA construct.

The vector may then include other elements, such as an origin of replication or a selector gene. These elements are well known in the art and can be incorporated therein using standard techniques. In one embodiment, the plasmid vector has the sequence of SEQ ID NO: 14, and (c) a sequence of (c). Alternatively, the construct may be included in a viral vector construct.

In certain embodiments, a nucleic acid construct of the invention may comprise two or more chimeric promoters as defined herein. Thus, the construct may comprise a plurality of chimeric promoters, in particular, the construct may have two, three, four, five or more chimeric promoters. Preferably, the chimeric promoters are each operably linked to a cloning site for insertion of a coding sequence. Thus, the construct may express two, three, four, five or more coding sequences. The coding sequence expressed may be any of the sequences described herein. In a preferred embodiment, the construct has two chimeric promoters, each of which is operably linked to a coding sequence. In particular, the two promoters may be transcribed independently of each other. In one embodiment, the promoters may transcribe toward each other. Thus, the promoters may be in opposite directions, or in some cases in the same direction.

In particular, constructs with two promoters can express the a and B subunits of ADP-ribosylating bacterial toxins, including any of those described herein, and preferably the LTA and B subunits. However, a construct with multiple promoters may express any combination of the coding sequences described herein. In a more preferred aspect, the construct with multiple promoters can express multiple influenza virus antigens, including combinations of any of the influenza virus antigens described herein. In another preferred aspect, the construct may express an influenza virus antigen, an immunogenic fragment thereof, or an immunogenic variant of both, and one or more of any of the coding sequences described herein.

Where the construct has multiple chimeric promoters, each promoter includes or is operably linked to any of the sequences mentioned herein. In a particularly preferred embodiment, the heterologous intron of one or more promoters may be the rat insulin gene intron a sequence. The one or more chimeric promoters may also preferably comprise the 5' UTR of HBV pre-S2. The one or more promoters may comprise a polyadenylation sequence of the rabbit beta globin gene.

In a preferred embodiment, the nucleic acid construct of the invention may comprise two chimeric promoter sequences, each operably linked to a cloning site for insertion of a coding sequence, wherein each chimeric promoter comprises:

(a) hCMV immediate early promoter sequence;

(c) a heterologous intron replacing the intron a region of the hCMV major immediate early gene.

Coding sequences operably linked to one chimeric promoter encode an LTA subunit and coding sequences operably linked to another promoter encode an LTB subunit. Thus, the construct is capable of expressing both subunits. Preferably:

-the heterologous intron of each promoter is the rat insulin gene intron a sequence;

-a sequence encoding each LT subunit is operably linked to the 5' UTR of HBV pre-S2; and/or

The LT coding sequence is operably linked to the polyadenylation sequence of the rabbit β -globin gene.

In a particularly preferred embodiment, one or more elements of vectors pPJV7563 and/or pjv1671 may be used in the nucleic acid construct of the invention. Functional fragments or variants of these elements may be used. In particular, any of the elements of pppjv 1671 are listed in table 3, functional variants of these elements, or functional fragments of both, may be used. Likewise, any of the elements of pppjv 7563 listed in example 5, functional variants of these elements, or functional fragments of both, may be used. Preferably, sequences of one or more of these elements of pPJV7563 and/or pjv1671 itself may be used. In another preferred embodiment, the corresponding elements of construct pml7789 may be used and the positions of these elements are specifically shown in figure 21 and the sequence listing provided herein. In a more preferred example, the corresponding elements of vectors pppjv 2012 and pPJV7788, specifically those shown in tables 6 and 8, can be used. Functional fragments and variants of the elements of the vectors pml7789, pPJV2012 and pjv7788 may also be used in the constructs of the invention.

In a preferred embodiment, the construct of the invention comprises:

(i) the sequence of vector pppjv 7563, SEQ ID No: 14, providing;

(ii) (ii) a sequence having 60% sequence identity to the sequence of (i),

in addition, a sequence encoding an influenza virus antigen, immunogenic fragment or immunogenic variant is inserted into the sequence of (i) or (ii) such that it is operably linked to the chimeric promoter. In a preferred embodiment, the coding sequence of the construct encodes an immunogenic fragment of the HA antigen, an immunogenic variant thereof, or both.

In a more preferred embodiment of the present invention, there is provided a construct wherein the vector comprises a nucleic acid sequence as set forth in SEQ ID No: 54, or a sequence having 60% sequence identity thereto, in a vector pppjv 1671. In another preferred embodiment, the vector comprises the sequence set forth as SEQ ID No: 59, or a sequence having 60% sequence identity thereto.

In a preferred example, the CMV promoter, untranslated leader, rat insulin intron a, untranslated leader, HBV enhancer and/or rabbit polyA sequences of pppjv 7563 and/or pPJV1671 or functional variants or fragments of these sequences may be used. In a particularly preferred embodiment, ppJV7563 and/or ppJV1671 of rat insulin intron A and/or HBV enhancer or functional fragments or variants of these sequences may be used. In particular, both rat insulin intron a and HBV enhancer are used or functional fragments or variants of these sequences are used. In another preferred example, the corresponding sequences of pml7789, pPJV2012, pjv7788, functional fragments of these sequences or variants of these sequences may be used.

In the case where the construct encodes several polypeptides, particularly antigens, specific sequences may be deleted to reduce the size of the construct. For example, the construct may lack an enhancer and/or an untranslated leader sequence, particularly when encoding three, four, five, or more antigens. These sequences are still present in other constructs encoding the same number of antigens.

In a particularly preferred embodiment, the vector pppjv 7563 can be used to clone a desired coding sequence, particularly a coding sequence for an antigen such as any of the antigens described herein. In a preferred example, the desired influenza virus antigen, fragment or variant thereof is cloned into a vector. In some cases pPJV7563 may be modified. For example, the kanamycin resistance gene can be replaced by another alternative gene, in particular another alternative selectable or screenable marker. Any of the nucleotide constructs of the invention may include a selectable marker. The pUC19 plasmid backbone of pPJV7563 can be modified or replaced with a different plasmid backbone. For example, a particular element of ppJV7563 may be replaced with any element that performs the same function, particularly a functional variant or fragment of the sequence in pJV 7563. In some cases, pPJV7563 may be modified by replacing a particular element with any of the elements described herein, which have the same function as the element to be replaced. Similar modifications can be made using pppjv 1671. Similar modifications can also be made to any of the vectors described herein, particularly to pPML7789, pPJV2012 and pPJV 7788. The coding sequences of pml7789, pPJV2012 and pjv7788 may be replaced by any of the coding sequences described herein. For pppjv 2012 and pPJV7788, one or both coding sequences of the vector may be replaced.

In a preferred embodiment, the nucleic acid construct of the invention may comprise:

(i) the sequence of vector pppjv 7563, SEQ ID No: 14, providing;

(ii) (ii) a sequence having 60% sequence identity to the sequence of (i),

in addition, the desired coding sequence is inserted such that it is operably linked to the chimeric promoter. In a preferred embodiment, the coding sequence encodes an antigen. In particular, a coding sequence encoding an influenza virus antigen, immunogenic fragment or immunogenic variant is inserted into the sequence of (i) or (ii) such that it is operably linked to a chimeric promoter.

This construct has 60% sequence identity to the sequence of (i), whether or not the inserted coding sequence is considered. The construct may have a sequence identity of any of the values mentioned herein, in particular at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%. In a preferred embodiment, the construct is pPJV7563 with an inserted coding sequence.

Other sequences may be added to the sequence of pPJV7563 to form other constructs of the invention. For example, one or more chimeric promoters of the invention may be added, as well as any of the sequences mentioned herein, operably linked to such a promoter. The cloning site of pppjv 7563 may be modified to clone different restriction fragments, particularly as the coding sequence for the different fragments. The same modifications can be made to any of the vectors described herein, and in particular to pPML7789, pPJV2012 and pPJV 7788.

In another preferred example, a construct of the invention may be made by replacing some or all of the coding sequence of pppjv 1671 encoding the antigen with a sequence encoding a different antigen. In addition, ppJV1671 may be modified by any of the modifications described above in relation to the portion of pPJV 7563. Such modifications may also be made to pPML 7789. The coding sequences of pPJV2012 and pPJV7788 may also be replaced by other desired coding sequences, especially those encoding antigens.

In other preferred examples, the constructs of the invention comprise:

-is provided as SEQ ID No: 54 or a sequence having 60% sequence identity thereto;

-is provided as SEQ ID No: 59 or a sequence having 60% sequence identity thereto;

-by SEQ ID No: 61 or a sequence having 60% sequence identity thereto, provided vector pppjv 2012;

-by SEQ ID No: 62 or a sequence having 60% sequence identity thereto.

In these examples, the construct may have any percentage of sequence identity described herein, particularly those described above in relation to the portions of pppJV 7563.

The constructs of the invention may comprise any of the elements listed in the tables herein, particularly those listed in tables 3, 6 and 8. Functional fragments of these sequences, as well as variants of these sequences and fragments thereof, may also be used. In a preferred example, if the carrier is an adjuvant carrier, one or more of the elements listed in tables 6 and/or 8 or fragments or variants of these sequences may be present.

The invention also provides a method of forming a construct of the invention, said method comprising inserting coding sequences for polypeptides, particularly antigens, into a vector of the invention lacking these sequences. The coding sequence may encode any of the antigens described herein. In a preferred embodiment, the present invention provides a method of inserting a selected coding sequence into one of ppJV7563, pJV1671, or a modified form of the vectors described herein. They may be inserted into pml7789, pPJV2012 and/or pPJV7788, especially pml 7789. In some cases, additional coding sequences may be inserted and/or existing coding sequences may be replaced with different coding sequences.

The invention also provides a promoter sequence and a coding sequence operably linked to the promoter, wherein the construct further comprises:

(a) A non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence and operably linked to the coding sequence and a promoter heterologous to the coding sequence; and/or

The plurality of elements of the carrier may be any of the elements described herein. In one example, the promoter sequence (i) is selected from the group consisting of an hCMV immediate early promoter sequence, a pseudorabies virus promoter sequence, and a rous sarcoma virus promoter sequence. In another example, the promoter is one of the chimeric promoters described herein.

In another example, the invention provides a purified, isolated chimeric promoter, wherein the chimeric promoter is any of the chimeric promoters defined herein.

The polynucleotide constructs of the invention are substantially free of or associated with cells or cellular material. It may be in a substantially isolated form, or in a substantially purified form, in each case generally comprising at least 90%, such as at least 95%, 98% or 99% of the polynucleotide or dry matter in the formulation.

The nucleic acid molecules of the invention can be delivered to a suitable host cell for expression of a polynucleotide operably linked to a promoter. Preferably, the host cell is a mammalian cell, in particular a human cell. Suitable methods for delivering nucleic acids into these cells are well known in the art and include, for example, dextran-mediated transfection, calcium phosphate precipitation, electroporation, and direct microinjection into the nucleus of the cell. Accordingly, the present invention provides a cell transformant having the vector of the present invention.

As described above, the nucleic acid coding sequence in the construct may encode a therapeutically relevant polypeptide. Thus, the constructs can be used for nucleic acid vaccination or gene therapy using standard gene delivery protocols. Suitable gene delivery methods are well known in the art and are described in detail below. The nucleic acid molecule can be delivered directly to the subject, or in vitro to cells from the subject, which are then reimplanted into the subject. In a preferred embodiment, the construct may be delivered directly to a subject, wherein the encoded polypeptide is an antigen, particularly an influenza antigen. Any of the delivery routes described herein may be used, particularly transdermal delivery. The adjuvant constructs of the invention may be administered to enhance an immune response to an antigen, particularly to an antigen expressed by the constructs of the invention.

The invention also provides the use of a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention or coated particles of the invention in the preparation of a medicament for nucleic acid vaccination. The medicament is a medicament for delivery by injection, transdermal particle delivery, inhalation delivery, topical delivery, oral delivery, intranasal delivery, or transmucosal delivery. In a preferred embodiment, the drug is delivered by needle-free injection.

For use in nucleic acid immunization or gene therapy, the nucleic acid construct can be prepared into conventional pharmaceutical preparations. Can be prepared using standard pharmacological formulation chemistry and procedures available to those of ordinary skill in the art. For example, a composition comprising one or more nucleic acid sequences (e.g., in the form of a suitable vector such as a DNA plasmid) can be combined with one or more pharmaceutically acceptable excipients or carriers to provide a liquid formulation. Thus also provided is a pharmaceutical composition comprising a nucleic acid construct of the invention and a pharmaceutically acceptable carrier or excipient. In a preferred embodiment, the pharmaceutical composition may comprise a plurality of constructs of the invention or a population of constructs of the invention, including any of the constructs described herein.

Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or carrier. These excipients, carriers and auxiliary substances are generally pharmaceutical agents which can be administered without undue toxicity and which, when used as a vaccine composition, do not elicit an immune response in the individual receiving the composition. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmacologically acceptable salts may also be included, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate and the like, organic acid salts such as acetate, propionate, malonate, benzoate and the like. Although not required, it is preferred that the formulation contain pharmaceutically acceptable excipients that act as stabilizers specifically for the peptide, protein or other molecule (if they are included in the composition). Examples of suitable carriers that also function as stabilizers for the peptide include, but are not limited to, pharmaceutical grades of glucose, sucrose, lactose, trehalose, mannitol, sorbitol, cellophane, dextran, and the like. Other suitable carriers also include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), and combinations thereof. Pharmaceutically acceptable excipients, carriers and auxiliary substances are described in great detail in REMINGTON' S PHARMACEUTICAL SCIENCES (Mark pub. co., n.j.1991), which is incorporated herein by reference.

Also included in the compositions are certain promoters of nucleic acid uptake and/or expression (transfection promoters), for example promoters such as bupivacaine, cardiotoxin and sucrose, and transfection facilitating carriers such as liposomal formulations or liposomal formulations commonly used for delivery of nucleic acid molecules. Anionic Liposomes and neutral Liposomes are widely available and are widely known for the delivery of nucleic acid molecules (see, e.g., Liposomes: A practical Approach, (1990) RPC New Ed., IRL Press). Cationic lipid formulations are also well known vehicles for delivering nucleic acid molecules. Suitable lipid formulations include those sold under the trade name Lipofectin^TMDOTMA (N- [1- (2, 3-dioleoyloxy) propyl) of (1)]N, N, N-trimethylaminichloride, N- [1- (2, 3-dioleyloxy) propyl]N, N, N-trimethyllamonium chloride) and DOTAP (1, 2-bis (oleoyloxy) -3- (trimethylammonium) propane, 1, 2-bis (dioxyloxy) -3- (trimethyllamonio) propane), see e.g. Felgner et al (1987) Proc. Natl. Acad. Sci.USA84：7413-7416；Malone et al.(1989)Proc.Natl.Acad.Sci.USA86: 6077-; U.S. Pat. Nos. 5,283,185 and 5,527,928, and International publication Nos. WO90/11092, WO91/15501, and WO 95/26356. These cationic lipids may preferably be used in combination with neutral lipids such as DOPE (dioleoylphosphatidylethanolamine). Other transfection-facilitating compositions that may be added to the above lipid or liposome formulations include spermine derivatives (see, e.g., international publication No. wo 93/18759) and membrane-penetrating compounds such as GALA, gramicidin (Gramicidine S) and cationic bile salts (see, e.g., international publication No. wo 93/19768).

Alternatively, the nucleic acid molecules of the invention may be entrapped, imbibed or bound to a particulate carrier. Suitable particulate carriers include those derived from polymethylmethacrylate polymers (polymethylmethacrylates), and PLG particles derived from poly (lactide) and poly (lactide-co-glycolide). See, e.g., Jeffery et al (1993) pharm. Res10: 362-368. Other particulate systems and polymers may also be used, such as polymers like polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules. In a preferred embodiment, the constructs of the invention are deposited on a carrier in the presence of a nucleic acid condensing agent and a metal ion chelating agent. Preferred coagulants include cationic polymers, especially polyamines, especially polyarginine or polylysine. In a preferred embodiment, the polyamine is (Arg)₄Or (Arg)₆. Reference may be made to the techniques described in WO2004/208560, which may be used to form the coated vectors of the present inventionAnd (3) granules.

Once formed, the compositions can be delivered to a subject by a variety of known routes and techniques. For example, the liquid preparation may be provided in the form of an injection solution, suspension or emulsion, and administered by parenteral injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection using a common needle and syringe or a liquid jet injection system. Liquid formulations may also be administered topically to the skin or mucosal tissue, or provided in the form of a fine spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.

Alternatively, the compositions may be administered in vitro, as is known to deliver and re-implant transformed cells into a subject (e.g., dextran-mediated transfection, calcium phosphate precipitation, electroporation, and direct microinjection into the nucleus of a cell).

The compositions are administered to a subject in an amount compatible with the agent, which amount is prophylactically and/or therapeutically effective. Suitable effective amounts will fall within a relatively broad range, but are readily determined by one of ordinary skill in the art through routine experimentation. For determining the required amount, the "Physicians desk reference" and the "pharmacological Basis of treatment" by Goodman and Gilman (the pharmacological Basis of therapeutics) are useful. For example, an effective dose of the polynucleotide is generally expected to range from about 0.001 to 1000. mu.g, preferably from 0.001 to 100. mu.g, more preferably from 0.01 to 10.0. mu.g. In certain examples, the dose may be 0.1 to 100 μ g, preferably 0.5 μ g to 25 μ g. When the agent is administered by needle-free injection, in some instances, the dose may be from 0.1 to 25 μ g, preferably from 0.5 μ g to 10 μ g, more preferably from 1 to 5 μ g. Specifically, the dose may be 4 μ g. In some cases, the dose may be administered by multiple needle-free injections, e.g., one, two, three, four, or five needle-free injections.

In some cases, the construct may be administered again after the first administration. Specifically, after the first immunization, the subject is then boosted. For example, the boosting can be a dose selected from any of the doses described herein. For example, the subject administration may be at least one week, two weeks, one month, two months, or six months after the first immunization.

In one example, a nucleic acid construct of the invention can be used in combination with another nucleic acid construct. In one example, the nucleic acid construct can be one of the constructs described herein that expresses an adjuvant, and the other construct can be a construct encoding one or more antigens. In a preferred embodiment, both constructs employ the chimeric promoters of the present invention. Where two or more substances herein are to be administered, they may in particular be administered separately, simultaneously or sequentially.

Where one construct expresses an adjuvant and the other construct expresses an antigen or antigens, the antigens may be particularly from HSV, HPV or hepatitis virus (particularly hepatitis b virus). The antigen may in particular be HSV ICP0, ICP4, ICP22 and/ICP 27 antigens, and preferably all four. In a particularly preferred example, the antigen is an influenza virus antigen, including any of the influenza virus antigens described herein, in particular HA, NA and/or M2, in particular HA and NA, in particular HA. In the case where these antigens are expressed, the adjuvant construct will specifically express LTA and/or LTB, especially both simultaneously. Any of the adjuvant constructs described herein may be used simultaneously, separately or sequentially with any of the constructs encoding the antigen.

Any two entities of the invention may be administered separately, simultaneously or sequentially. The two constructs may be administered separately, simultaneously or sequentially. The two constructs may be administered in the same composition or in different compositions. In particular, where one construct has an adjuvant effect, both constructs will be delivered, whereby an adjuvant effect is observed, namely: administration of an adjuvant with an antigen results in an enhanced and/or prolonged immune response compared to administration of the adjuvant without the antigen. In a preferred embodiment, the two constructs are delivered in the same composition, preferably on the same vector particle. In other cases, vector particles carrying one construct may be mixed with vector particles carrying another construct. For any combination of the various constructs described herein, any such dosing regimen may be used.

In a preferred embodiment, the nucleic acid construct of the invention is delivered to a target cell using particle-mediated delivery techniques. Particle-mediated methods for delivering nucleic acid agents are well known in the art.

Thus, in a preferred embodiment, the invention provides a coated particle comprising a vector particle coated with a nucleic acid construct of the invention or a population of nucleic acid constructs of the invention. In particular, the coated particles are suitable for delivery from a particle-mediated delivery device.

Particles for use in particle-mediated delivery systems can be formed by coating the nucleic acid molecules of the invention onto a carrier particle (e.g., a core carrier) using a variety of techniques well known in the art. The carrier particles are selected from a group of materials having a suitable density within the particle size range that is generally useful for intracellular delivery from a particle-mediated delivery device. Typically, the carrier particles have a diameter of from 0.1 to 5 μm, such as from 0.5 to 3 μm, preferably from 1 to 2 μm. In some cases, the particles have a diameter of 1 to 3 μm. The optimal vector particle size will of course depend on the diameter of the target cell.

Typically, the support particles are selected from inert metals. The metals are inert because they are not physiologically active. For the purposes of the present invention, it is possible to use, for example, iron, cobalt, nickel, copper, silver, cadmium, hafnium, tantalum, tungsten, platinum, gold and stainless steel, in particular tungsten, gold, platinum and iridium support particles. Tungsten and gold particles are preferred. Thus, in a preferred embodiment, the present invention provides coated support particles of gold or tungsten. Tungsten particles having an average diameter of 0.5 to 2.0 μm are readily available. Although such particles have an optimal density for use in particle-accelerated delivery methods and offer the potential for efficient coating with DNA, tungsten may be toxic to certain cell types. Gold particles or microcrystalline gold (e.g., gold powder a1570, available from Engelhard corp., East Newark, NJ) have also been found to be useful in the present process. Gold particles are uniform in size (1-3 μm in size, obtained from Alpha Chemicals, or from Degussa, South Plainfield, NJ, particle size in a range including 0.95 μm) and have reduced toxicity. Microcrystalline gold has a different particle size distribution, typically in the range of 0.1-5 μm. However, the irregular surface area of microcrystalline gold provides the possibility of efficient coating with nucleic acids.

Various methods of coating or depositing DNA or RNA on gold or tungsten particles are known and have been described. Most of these methods generally involve mixing a predetermined amount of gold or tungsten with plasmid DNA, CaCl₂And spermidine. The resulting solution was stirred at all times during the coating to ensure homogeneity of the reaction mixture. Following nucleic acid deposition, the coated particles may be transferred to a suitable membrane and dried prior to use, and then coated onto the surface of a sample module or cassette or loaded into a delivery cassette particularly for use in a particle-mediated delivery device.

Alternatively, the polynucleotides of the invention may be prepared as microparticle compositions. Prepared using standard pharmacological formulation chemistry described above. For example, the polynucleotide may be combined with one or more pharmaceutically acceptable excipients or carriers to provide a suitable composition. The resulting composition is then prepared into particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilization), spray-freeze drying, spray coating, precipitation, supercritical fluid particle preparation, and the like. The resulting granules can be densified, if desired, using the techniques described in International publication No. WO97/48485, which is incorporated herein by reference.

These methods can be used to obtain nucleic acid particles in the size range of about 0.01 to about 250 μm, preferably about 10 to about 150 μm, and most preferably about 20 to about 60 μm; and the particle density ranges from about 0.1 to about 25g/cm³Bulk density of about 0.5 to about 3.0g/cm³Or greater.

Once formed, the nucleic acid molecule-containing particles can be packaged into a unit-dose or multi-unit-dose container. Accordingly, the present invention also provides a dosage container for a particle-mediated delivery device comprising a coated particle of the present invention. The container may comprise a sealed, tight container containing a suitable amount of the particles. The particles can be packaged as a sterile formulation, and thus, the hermetically sealed container can be specifically designed to maintain the sterility of the formulation prior to delivery to a subject. The container is preferably suitable for direct use with a particle-mediated delivery device. Typically, the container is in the form of a capsule, foil pouches, sachets, etc. The particle delivery device containing the appropriate dose of particles may be provided in a pre-loaded form. The pre-load device may then also be pre-packaged in a tightly closed container.

The container in which the particles are packaged may be further marked to identify the composition and provide relevant dosage information. In addition, the container may be labeled with a label in a form prescribed by a governmental agency, such as the food and drug administration, wherein the label indicates that the manufacture, use or sale of the human nucleic acid preparation contained therein is permitted by the federal legal authorities.

The invention also provides particle-mediated delivery devices loaded with the coated particles of the invention. Preferably, the delivery device is a needleless syringe. Particle accelerating devices suitable for use in particle-mediated delivery devices are well known in the art. For example, existing particle gun devices use explosive, electrical, or gaseous release methods to propel coated carrier particles to target cells. The coated carrier particles may be releasably attached to a removable carrier sheet or removably attached to a surface through which the gas stream passes, lifting the particles off the surface and accelerating to the target. An example of a gas release device is described in U.S. patent No.5,204,253. An explosion-type device is described in U.S. Pat. No.4,945,050. One example of an electrical discharge device suitable for use herein is described in U.S. Pat. No.5,120,657. Another type of electrical discharge device is described in U.S. patent No.5,149,655. The disclosures of all of these patents are incorporated herein by reference in their entirety.

The particles may also be administered using a needleless injection device, such as that described in U.S. Pat. No.5,630,796 to Bellhouse et al ("the PowderJect^®needle-free system device ") and international publication nos. WO 94/24263, WO 96/04947, WO 96/12513 and WO 96/20022, all of which are incorporated herein by reference.

A device such as that described in us patent No.5,630,796 may be a pen-like device comprising, in order from top to bottom in linear sequence, a gas cartridge, a particle cassette or a particle bag, and an ultrasonic nozzle connected to a muffler. The particles may be provided in a suitable container such as a box or the like formed from two breakable polymeric films which are heat sealed into a gasket-like spacer to form individual closed units. The membrane material may be selected to obtain a particular opening pattern and burst pressure, which indicates the conditions under which the supersonic flow is initiated.

In operation, the device is caused to release compressed gas from the gas cartridge into an expansion chamber within the device. The released gas contacts the particle cassette and, upon reaching sufficient pressure, suddenly breaks through the cassette membrane to direct the particles into the ultrasonic nozzle for subsequent delivery. The nozzle is designed to achieve a specific gas velocity and flow pattern to deliver a quantity of particles to a target surface in a predetermined area. Mufflers are used to attenuate the noise generated by ultrasonic airflow.

The delivery system described in international publication No. wo 96/20022 also uses the energy of a compressed gas source to accelerate and deliver the powdered composition. However, it differs from the system of us patent No.5,630,796 in that it uses shock waves instead of a gas stream to accelerate the particles. More specifically, the rise in instantaneous pressure created by the shock wave behind the elastic dome strikes the back of the dome, causing a sudden eversion of the elastic dome in the direction along the target surface. This snap-out action emits the powdered composition (which is outside the dome) at a sufficient velocity and thus with sufficient momentum to penetrate the target tissue, such as the oral mucosal tissue. The powdered composition is released immediately upon full eversion of the dome. The dome also contains the high pressure air flow completely so it does not come into contact with tissue. The system is quiet in nature since gas is not released during the delivery operation. Such a design may be used in other closed or otherwise very sensitive applications, for example to deliver particles to minimally damaged surgical sites.

The particles can be delivered directly to the subject in vivo, or to cells from the subject in vitro, and the transformed cells then reimplanted into the subject. For delivery in vivo, injection of the particles is typically subcutaneous, epidermal, intradermal, intramucosal (e.g., nasal, rectal, and/or vaginal), intraperitoneal, intravenous, buccal, or intramuscular. Preferably, to terminally differentiated cells; however, the particles can also be delivered to stem cells of undifferentiated, or partially differentiated cells such as blood and skin fibroblasts. Most preferably, to skin epidermal cells.

The particles are administered to a subject in a manner compatible with the dosage formulation and in an amount that is prophylactically and/or therapeutically effective. A "therapeutically effective dose" of the microparticle composition is sufficient to treat or prevent the symptoms of a disease or disorder and will fall within a relatively wide range that can be determined using conventional methods. Generally, the particles are delivered in an amount of 0.001 to 1000. mu.g, more preferably 0.01 to 10.0. mu.g, of nucleic acid per dose. However, the precise dosage required will vary with the age and general condition of the individual being treated, as well as the particular nucleic acid sequence selected and other factors. The appropriate effective amount can be readily determined by clinical examination. The "physician pharmacy reference book" and the "pharmacological basis of treatment" by Goodman and Gilman are useful in determining the required amount.

Analysis of

Differential expression analysis

Appropriate element utility assays can determine the effect of an element on polypeptide expression. In a preferred example, the polypeptide may be a fragment of an influenza virus antigen, a variant thereof, or both. The basis for comparison of the utility of the test elements is the "basic vector", which generally (unless otherwise mentioned) refers to a plasmid containing the hCMV promoter, hCMV exon 1, 9 bases of hCMV exon 2, the 5' UTR of HBV preS2 and the rabbit β -globin polyadenylation region, which is located where the expression of the driving coding sequence is located. Typically, the basic vector is pJV7384, pJV7401, pJV7450 or pJV 7533. In certain examples, any of the vectors described herein having the above elements can be used as the primary vector. In one example, pPJV1671 is used as the base vector. pppjv 2012, pPJV7788 and pPML7789 can also be used as basic vectors.

Adding a heterologous intron and a 3 'UTR into a basic vector, or introducing a promoter sequence, an exon, a 5' UTR and a polyA locus into the basic vector to form an expression vector to be detected. Elements of any of the vectors described herein can be introduced with the test sequence and compared to the base sequence. Thus, functional variants or fragments may be detected.

The basic vector and the test vector are transformed into a suitable host cell and the cell is analyzed for the level of polypeptide expression. Preferably, mammalian host cells are used. Suitable cells include mammalian HEK 293T, CHO, HeLa, BHK, 3T3 or COS cells. In certain examples, SSC15 or B16 cells can be used.

Generally, the functional element will produce expression comparable to, e.g., at least the same or higher than, the basic vector. Preferably, expression is in more than one cell type and detected using more than one coding sequence. In certain examples, the functional element may cause slightly lower expression than the base vector, e.g., at least 25%, particularly at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and yet more preferably at least 90% of the expression of the base vector. In general, if a variant or fragment of a particular element exhibits these levels of expression, it is still considered to represent a functional variant or fragment of the particular element. However, it is preferred that functional variants and fragments will result in higher expression than the basic vectors described herein. For example, the percent increase can be any of the percentages described above.

Suitable experimental methods are provided, for example, in examples 1 to 18 below.

Differential immunogenicity assays

If the polypeptide to be expressed is an antigen, a further test is carried out to identify functional construct elements or particularly preferred construct elements. In particular, such a test may be performed wherein the antigen is, for example, an influenza virus antigen, a fragment thereof, or a variant of both. In this assay, the effect of the element on the immune response is measured after delivery of the expression vector to the test organism. Antibody levels against antigens are the simplest way to judge the immune response. Different groups of mice were inoculated with the basic vector or test vector constructed as above. Sera were collected after the appropriate time and analyzed for antibody levels.

The experiment was performed in duplicate and the antibody levels obtained in all groups of duplicate experiments were plotted. In both experiments, the functional element will produce at least the same or higher antibody titer against a particular antigen compared to the basic vector. Preferably, the result can be observed using more than one antigen, indicating the practical range of elements in the expression series. In certain examples, the functional element may elicit a slightly lower antibody titer than the base vector, e.g., at least 25%, particularly at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, still more preferably at least 90% of the antibody titer observed in the base vector. In general, if a variant or fragment of a particular element exhibits such an antibody titer, it is still considered to represent a functional variant or fragment of the particular element. However, it is preferred that functional variants and fragments will result in higher titers than the basic vectors described herein. For example, the percent increase can be any of the percentages described above.

Adjuvant carriers can also be evaluated by comparing test adjuvant carriers to standard adjuvants, all given the same antigen. The adjuvant effect of both carriers was compared using antigen alone as a control. Any of the adjuvant carriers described herein can be used as standards. The percentage of increase or decrease in adjuvant effect may be any of these levels described herein.

A suitable experimental method is provided, for example, in example 14 below. Suitable experimental protocols are also described in examples 15 to 18 below.

C.Experiment of

The following are examples of specific embodiments for practicing the present invention. These examples are provided for illustration only and are not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should, of course, be allowed for.

Method

Standard PCR conditions

The standard PCR conditions used to construct the vector are as follows: containing 1.5mM MgCl₂1 XPCR core buffer (Promega Corporation, Madison, Wis.), 0.400. mu.M for each primer, 200. mu.M for each dNTP (USB, Inc, Cleveland, OH), 2.5. mu.Taq polymerase (Promega Corporation, Madison, Wis.), 1.0ng template DNA, water to 100. mu.l, and covering with mineral oil (Aldrich Chemical, Inc, Milwaukee, Wis.). A PTC-200 thermocycler (thermocycler) (MJ Research, inc. waltham, MA) is programmed to perform the following procedure: 95 ℃ X4 ', 30 cycles (95 ℃ X1 '/55 ℃ X1 ' 15 "/72 ℃ X1 '), 72 ℃ X10 ', 4 ℃ hold). Prior to cleavage with restriction endonuclease (New England Biolabs, Beverly, MA), the amplified product was removed from the PCR reaction using QIAquick PCR purification kit (Qiagen Inc, Valencia, CA).

All PCR products were sequenced after cloning to determine the fidelity of amplification.

Example 1 construction of hepatitis B Virus surface antigen (HBsAg) vector series

A number of plasmid expression vectors expressing HBsAg were constructed.

Raw material

(i)pWRG7128(Roy，M，et.al.Vaccine(2001)19: 764-778) containing the hCMV immediate early promoter sequence, the first exon, the first intron and part of the second exon of the hCMV major immediate early gene, the HBsAg coding sequence with flanking regions (sequences derived from the HBV preS 25 'UTR and 3' post-transcriptional response element) and the bovine growth hormone polyadenylation region (BGHpA).

(ii) pJV7284, a derivative of pWRG7128, which replaces BGHpA with the rabbit globin polyadenylation region (RBGpA).

(a) pPJV7384(CMV (intron-free), HBV preS 25' UTR and RBGpA)

pWRG7128, Sal 1 and BamH 1 splice were amplified with JF93(SEQ ID NO: 15) and F110(SEQ ID NO: 16) using standard conditions to isolate an insert containing the CMV promoter, exon 1 and part of the exon 2 sequence. BamH 1 and BstX1 cleave pAM6(ATCC, Mannassas, Va.) to isolate an insert containing the HBsAg 5' UTR and approximately 70% of the HBsAg coding region. Sal 1 and BstX1 cut pJV7284 to form a vector fragment with these two inserts ligated into it, resulting in pJV 7293.

pWRG7128 was PCR amplified with primers GW1(SEQ ID NO: 17) and JF254(SEQ ID NO: 18) and cleaved with BstX 1 and Bgl2 to isolate the insert containing the 3' end of the HBsAg coding region. BstX 1 and Bgl2 cut pPJV7293 to form a vector fragment with the insert ligated into it, resulting in vector pPJV 7384.

(b) pPJV7382(CMV (no intron), HBsAg 3 'UTR, HBV preS 25' UTR and RBGpA)

Xho 1 and Xba 1 cut pPJV7293 to form an insert containing the CMV promoter/exon and 5 '-UTR with the 5' end of the HBsAg coding sequence. Xba 1 and Bcl 1 cut pWRG7128 to form an insert containing most of the HBsAg coding sequence and its 3' -UTR. Xho 1 and Bgl2 cut pPJV7284 to form a vector fragment with both inserts ligated into it, resulting in vector pjv 7382.

(c) pPPJV7389(CMV (RIA)), HBsAg 3 'UTR, HBV preS 25' UTR and RBGpA)

Rat insulin intron A (RIA) was PCR amplified from plasmid p 5' rlns (unknown origin) using primers GW150(SEQ ID NO: 19) and JF255(SEQ ID NO: 20). BamH 1 cleaves the PCR product and inserts BamH 1 linearized pppjv 7382, yielding pPJV 7389.

(d) pPJV7387(CMV (RIA)), HBV preS 25' UTR and RBGpA)

BstX 1 and EcoR1 cleave pPJV7384 to form an insert containing the 3' end of the HBsAg coding region and RBGpA. BstX 1 and EcoR1 cleave pPJV7389 to form a vector fragment into which this insert is ligated, resulting in vector pPJV 7387.

Example 2 construction of the herpes simplex Virus glycoprotein D antigen (HSVgD) vector series

A number of plasmid expression vectors expressing HSVgD were constructed.

Raw material

(a) pPJV7334, a derivative of pWRG7284(pPJV 7284), the HBsAg coding sequence was replaced by an in-frame Nhe 1 immediately downstream of the ATG start codon, followed by a stuffer with BamH1, immediately 5' to the HBC Enh (enhancer).

(b) pWRG7202, a derivative of pGem3Z (Promega) containing a stuffer fragment that provides the possibility of fusing the coding sequence to the human Tissue Plasminogen Activator (TPA) signal peptide downstream of the Nhe 1 site.

(a) pPJV7392(CMV (Natural intron), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

The coding region of HSV2gD was PCR amplified from a viral DNA stock (AdcancedBiotech, Inc, Columbia, Md.) using primers DS1(SEQ ID NO: 21) and DA1(SEQ ID NO: 22) and cleaved with Nhe 1 and EcoR1 to form an insert. Nhe 1 and EcoR1 cleave pWRG7202 to form a vector fragment in which the insert is ligated, resulting in vector pPJV 7391.

Nhe 1 and Bgl 2 cleave pppjv 7391 to form an insert containing the coding sequence of HSV2 gD. Nhe 1 and BamH 1 cleave pPJV7334 to form a vector fragment into which the insert is ligated, resulting in vector pjv 7392. The vector consisted of the following expression elements: hCMV immediate early promoter sequence, the first exon, the first intron and part of the second exon of the hCMV major immediate early gene, the 5 '-UTR of HBsAg, the coding sequence of HSV2 gD gene, the 3' -UTR of HBsAg and RBGpA.

(b) pPJV7399(CMV (without intron), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

An intron-free pPJV7392 was constructed as follows. pPJV7384 was cut with HindIII and Nde 1 to isolate an insert containing the 5' end of the kanamycin resistance gene and the CMV promoter. Nde 1 and Ssp1 cut pppjv 7384 to isolate the insert containing the CMV promoter 3 ' end, CMV exon 1/2 and the 5 ' end of the HBsAg5 ' -UTR. This insert was inserted into pppjv 7392, and the Hind 3-sssp 1 fragment was removed, yielding pppjv 7399.

(c) pPJV7400(CMV (RIA), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

Pppjv 7392 in the RIA form was constructed as follows. pPJV7384 was cut with HindIII and Nde 1 to isolate an insert containing the 5' end of the kanamycin resistance gene and the CMV promoter. Nde 1 and Ssp1 cut pppjv 7387 to isolate the insert containing the CMV promoter 3 ' end, CMV exon 1/2 (part), RIA and HBsAg5 ' -UTR 5 ' end. This insert was inserted into pppjv 7392 and the Hind3-Ssp 1 fragment was removed, yielding pppjv 7400.

(d) pPJV7401(CMV (without intron), HBsAg 5' UTR and RBGpA)

pJV7399 without 3' -UTR was constructed as follows. Bsp 120I and Bgl2 cut pPJV7391 to isolate the insert containing the 3' end of the HSV2 gD gene. Bgl2 and EcoR1 cut pJV7284 to isolate the RBGpA signal. These inserts were inserted into ppJV7399 and the Bsp 120I-EcoR1 fragment was removed to give pPJV 7401.

(e) pPJV7402(CMV (RIA), HBsAg 5' UTR and RBGpA)

The 3' -UTR-free pPJV7400 was constructed as follows. Bsp 1201 and Bgl2 cleave pPJV7391 to isolate the insert containing the 3' end of the HSV2 gD gene. Bgl2 and EcoR1 cut pPJV7284 to isolate the RBGpA signal. These inserts were ligated into ppJV7400 and the Bsp 120I-EcoR1 fragment was removed to give pPJV 7402.

Example 3 construction of Flu M2 antigen vector series

(a) pPJV7450(CMV (without intron), HBsAg 5' UTR and RBGpA)

The Flu M2 coding region was PCR amplified from plasmid pFL-M2(Joel Haynes, pPJV) using primers JF301(SEQ ID NO: 23) and JF302(SEQ ID NO: 24) and cleaved with Nhe 1 and Bgl2 to form an insert. Nhe 1 and Bgl2 cut pppjv 7401 to form a vector fragment with the insert ligated into it, resulting in pjv 7450.

(b) pPJV7452(CMV (without intron), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

The 3' UTR fragment was PCR amplified from pPJV7389 using primers JF84(SEQ ID NO: 25) and JF225(SEQ ID NO: 26), Bsp120I cleaved, T4 DNA polymerase filled, and Bgl2 linker (cat #1036, New England Biolabs) ligated. This fragment was then cleaved with Bgl2 and EcoR1 to isolate the insert containing the HBsAg 3' UTR and RBGpA regions. Bgl2 and EcoR1 cut pPJV7450 to form a vector fragment in which the insert was ligated, resulting in pPJV 7452.

(c) pPJV7458(CMV (RIA), HBsAg 5' UTR and RBGpA)

pppJV 7450 containing the RIA form was constructed as follows: BamH 1 cleaves pPJV7389 to isolate RIA-containing inserts. BamH 1 cleaves pPJV7450 to form a vector fragment into which the insert is ligated, resulting in pPJV 7458.

(d) pPJV7468(CMV (RIA), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

pPJV7458 containing HBsAg 3' UTR was constructed as follows. Bgl2 and EcoR1 cut pPJV7452 to make an insert containing HBsAg 3' UTR and RBGpA. Bgl2 and EcoR1 cleave pPJV7458 to form a vector fragment into which the insert is ligated, resulting in pPJV 7468.

Example 4 construction of the beta-gal vector series

(a) pPJV7488(CMV (without intron), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

CMV- β (Clontech) was PCR amplified using primers JF335(SEQ ID NO: 27) and JF336(SEQ ID NO: 28) and cleaved with Nhe 1 and Bgl 2 to isolate the insert encoding β -galactosidase. Nhe 1 and Bgl 2 cut pppjv 7452 to form a vector fragment with the insert ligated into it, resulting in pjv 7488.

(b) pPJV7533(CMV (without intron), HBsAg 5' UTR and RBGpA)

Bgl 2 and EcoR1 cleave pPJV7450 to isolate the insert containing RBGpA. Bgl 2 and EcoR1 cleave pPJV7488 to form a vector fragment into which the insert was ligated, resulting in pPJV 7533.

(c) pPJV7551(CMV (RIA/Nhel), HBsAg 3 'UTR, HBsAg 5' UTR and RBGpA)

Xho 1 and BamH 1 cut pPJV7530 (see example 5) to isolate the insert containing the CMV promoter through RIA. Xho 1 and BamH 1 cleave pppjv 7488 to form a vector fragment into which the insert is ligated, resulting in pPJV 7551.

(d) pPJV7552(CMV (RIA/Nhel), HBsAg 5' UTR and RBGpA)

Xho 1 and BamH 1 cut pPJV7530 to isolate the insert containing the CMV promoter crossing over to RIA. Xho 1 and BamH 1 cleave pppjv 7533 to form a vector fragment into which the insert is ligated, resulting in pjv 7552.

Example 5 construction of pPJV expression vector (pPJV7563)

(a)pPJV7496

pPJV7389 was amplified using primers JF357(SEQ ID NO: 29) and JF365(SEQ ID NO: 30) pCR, blunt-ended by treatment with T4 DNA polymerase, and Sal 1 cleaved to isolate the insert encoding the kanamycin resistance gene. Ava 1 cleavage pPJV7389, T4 DNA polymerase treatment blunted the termini, and Sal 1 cleavage to isolate the vector fragment with the insert ligated thereto, resulting in pPJV 7496.

(b)pPJV7530

pPJV7389, Bgl2 and BamH1 cleavage were PCR amplified using primers JF393(SEQ ID NO: 31) and JF406(SEQ ID NO: 32) to isolate an insert containing RIA (deletion of the internal Nhe1 site). BamH1 cleaves pPJV7496 to make a vector fragment into which the insert is ligated, resulting in pPJV 7530.

(c)pPJV7549

BamH1 and EcoR5 cleave pPJV7468 to isolate an insert containing M2 and partial HBV 3' ENH. BamH1 and EcoR5 cut pPJV7530 to prepare a vector fragment into which the insert was ligated, resulting in pPJV 7549.

(d)pPJV7563

The primers JF256(SEQ ID NO: 33) and JF257(SEQ ID NO: 34) were annealed to prepare an insert composed of a multiple cloning site. Nhe1 and Bgl2 cut pPJV7549 to prepare a vector fragment with the insert ligated therein, resulting in pjv 7563. FIG. 12 shows a pPJV7563 plasmid map. FIG. 13 shows the base composition of plasmid pPJV 7563. The components and their positions in plasmid pppjv 7563 are shown below:

1-44 transposon 903 sequence

45-860 kanamycin resistance coding sequence from transposon 903

861-896 transposon 903 sequence

897-902 Sal1 site

903-1587 CMV promoter

1588-1718 untranslated leader sequence from CMV immediate early Gene

1719 fusion of BamH1 and Bgl11 restriction enzymes

1725 1857 rat insulin intron A

1858-1863BamH1 site

1864-1984 HBV surface antigen 5' untranslated leader sequence

1985-1993 Synthesis of the initiation codon/Nhe 1 cloning site

1994-2011 synthetic cloning site

2012 + 2544 HBV enhancer

2545 2555 Primary vector sequence. No hits in NCBI database

2556 + 2686 Rabbit beta-globin polyadenylation region

2687-3759 pUC19 vector sequence

Example 6 use of human secreted alkaline phosphatase (SEAP) and human IgG Fc fragment (hFc) Construction of Signal peptide expression vector series for model antigens

(i) pPJV7507 (hTPPasp and SEAP)

pSEAP-basic (Clontech), Nhe1 and Bgl 2 cleavages were PCR amplified using primers JF320(SEQ ID NO: 35) and JF321(SEQ ID NO: 36) to isolate an insert consisting of a human SEAP fragment. Nhe1 and Bgl 2 cut pppjv 7079(Macklin, et al.) to make a vector fragment into which the insert was ligated, resulting in pjv 7507.

(ii) pPJV7508 (hTPPasp and hFc)

Human DNA was PCR amplified using primers JF386(SEQ ID NO: 37) and FcAS (SEQ ID NO: 38) and then cleaved with Nhe 1 and Bgl 2 to isolate an insert consisting of a human IgG Fc fragment. Nhe 1 and Bgl 2 cut pPJV7079 to prepare a vector fragment with the insert ligated thereto, resulting in pjv 7508.

(iii) Preparation of the coding sequence of the aprotinin Signal peptide

Synthetic oligonucleotide JF354(SEQ ID NO: 39) was PCR amplified using primers JF355(SEQ ID NO: 40) and JF356(SEQ ID NO: 41) to form the coding sequence for the aprotinin signal peptide.

(iv) Preparation of tobacco extensin signal peptide coding sequence

The synthetic oligonucleotide JF348(SEQ ID NO: 42) was PCR amplified using primers JF349(SEQ ID NO: 43) and JF350(SEQ ID NO: 44) to form the coding sequence for the tobacco extensin signal peptide.

(v) Preparation of signal peptide coding sequence of chicken lysozyme

The synthetic oligonucleotide JF351(SEQ ID NO: 45) was PCR amplified using primers JF352(SEQ ID NO: 46) and JF353(SEQ ID NO: 47) to form the coding sequence for the chicken lysozyme signal peptide.

(a) Flu M2 antigen signal peptide series

pPJV7499(CMV (without intron), HbsAg 5' UTR, RBGpA, aprotinin signal peptide)

pPJV7497(CMV (without intron), HbsAg 5' UTR, RBGpA, tobacco extensin signal peptide)

pPJV7500(CMV (without intron), HbsAg 5' UTR, RBGpA, chicken lysozyme signal peptide)

SPe 1 and Nhe 1 cleave the signal peptide coding sequence to isolate the insert. Nhe 1 cleaves pPJV7450 to prepare a vector fragment into which the insert was ligated, yielding pPJV7499 (aprotinin), pjv7497 (tobacco extensin) and pjv7500 (chicken lysozyme).

(b) SEAP signal peptide series

pPJV7513(CMV (without intron), HbsAg 5' UTR, RBGpA, aprotinin signal peptide)

pPJV7512(CMV (without intron), HbsAg 5' UTR, RBGpA, tobacco extensin signal peptide)

pPJV7510(CMV (without intron), HbsAg 5' UTR, RBGpA, chicken lysozyme signal peptide)

Xho 1 and Nhe 1 cut pppjv 7499, 7497 and 7500 to isolate an insert consisting of the CMV promoter which crosses the signal peptide coding sequence of the plasmid. Xho 1 and Nhe 1 cut pPJV7507 to prepare a vector fragment in which the insert was ligated, yielding pjv7513 (aprotinin), pjv7512 (tobacco extensin) and pjv7510 (chicken lysozyme).

(c) hFc signal peptide series

pPJV7524(CMV (without intron), HbsAg 5' UTR, RBGpA, aprotinin signal peptide)

pPJV7525(CMV (without intron), HbsAg 5' UTR, RBGpA, tobacco extensin signal peptide)

pPJV7526(CMV (without intron), HbsAg 5' UTR, RBGpA, chicken lysozyme signal peptide)

Xho 1 and Nhe1 cut pppjv 7499, 7497 and 7500 to isolate an insert consisting of the CMV promoter which crosses the signal peptide coding sequence of the plasmid. Xho 1 and Nhe1 cut pPJV7508 to prepare a vector fragment in which the insert was ligated, yielding pjv7524 (aprotinin), pjv7525 (tobacco extensin) and pjv7526 (chicken lysozyme).

Example 7 construction of human secreted alkaline phosphatase (SEAP) series

(a) pPJV7531(CMV (without intron), HbsAg 5' UTR, RBGpA, chicken lysozyme signal peptide)

Sal 1 and Bgl 2 cut pPJV7510 to isolate the insert containing the CMV promoter through the lysozyme signal peptide. Sal 1 and Bgl 2 cut pPJV7450 to form a vector fragment with the insert ligated therein, resulting in pPJV 7531.

(b) pPJV7554(CMV (RIA/Nhe1), HBsAg 5' UTR, RBGpA, chicken lysozyme signal peptide)

Xho 1 and BamH 1 cut pPJV7530 to isolate the insert containing the CMV promoter through RIA. Xho 1 and BamH 1 cleave pppjv 7531 to form a vector fragment into which the insert is ligated, resulting in pjv 7554.

(c) pPJV7568(CMV (intron-free), HBsAg3 'UTR, HBsAg 5' UTR, RBGpA, BsA, Chicken lysozyme signal peptide)

Bgl 2 and EcoR 1 cut pPJV7563 to isolate the insert containing HBV 3' UTR and RBGpA. Bgl 2 and EcoR 1 cleave pPJV7531 to form a vector fragment into which the insert is ligated, resulting in pPJV 7568.

(d)pPJV7572(CMV(RIA/Nhel)、HBsAg 3’UTR、HBsAg5’UTR、RBGpA、 Chicken lysozyme signal peptide)

Bgl 2 and EcoR 1 cut pPJV7563 to isolate the insert containing HBV 3' UTR and RBGpA. Bgl 2 and EcoR 1 cut pPJV7554 to form a vector fragment into which the insert was ligated, resulting in pPJV 7572.

Example 8 construction of beta-gal and HBsAg Using Chicken Keratin and Chicken Heart actin introns Carrier

(a) pPJV7557 (. beta. -gal, CMV (cA intron), HbsAg3 'UTR, HbsAg 5' UTR and RBGpA)

chicken DNA was PCR amplified using primers JF430(SEQ ID NO: 48) and JF442(SEQ ID NO: 49), and Bgl 2 and BamH 1 were cleaved to isolate an insert consisting of the intron and flanking exon sequences of chicken heart actin. BamH 1 cleaves pPJV7488 to make a vector fragment into which the insert is ligated, resulting in pPJV 7557.

(b) pPJV7558 (. beta. -gal, CMV (cK intron), HbsAg3 'UTR, HbsAg 5' UTR and RBGpA)

Chicken DNA was PCR amplified using primers JF421(SEQ ID NO: 50) and JF444(SEQ ID NO: 51), and Bgl 2 and BamH 1 were cleaved to isolate an insert consisting of the intron and flanking exon sequences of the chicken keratin gene. BamH 1 cleaves pPJV7488 to make a vector fragment into which the insert is ligated, resulting in pPJV 7558.

(c) pPJV7578(HBsAg, CMV (cA intron), HbsAg3 'UTR, HbsAg 5' UTR and RBGpA)

sal 1 and BamH 1 cut pPJV7557 to isolate the insert consisting of the CMV promoter through the intron region. Sal 1 and BamH 1 cut pPJV7496 to prepare a vector fragment into which the insert was ligated, resulting in pPJV 7558.

(d) pPJV7579(HBsAg, CMV (cA intron), HbsAg3 'UTR, HbsAg 5' UTR and RBGpA)

sal 1 and BamH 1 cut pPJV7558 to isolate the insert consisting of the CMV promoter through the intron region. Sal 1 and BamH 1 cut pPJV7496 to prepare a vector fragment into which the insert was ligated, resulting in pPJV 7579.

Example 9 in vitro analysis of HBsAg vector series antigen expression

On the first day, SCC15(ATCC) or B16 (unknown in origin, obtained from ATCC) cells were seeded at 20% -40% confluence (confluency) into 6-well tissue culture plates and grown overnight in an incubator. The host cells were propagated in the culture medium recommended by the ATCC.

On the next day, the transfection reaction was performed. For each vector to be tested, 20. mu.l Lipofectin was added^®Reagent (Life Technologies Inc, Grand Island, NY) was added to 180. mu.l Optimem^®Culture medium (Life Technologies Inc, Grand Island, NY) and incubation at room temperature for 45 min. For each vector to be tested, at 40min, 2. mu.g of vector and 200. mu.l of Optimem^®And (4) mixing. At 45min, the vector and Lipofection were added^®The solutions were mixed together and allowed to stand at room temperature for an additional 10 minutes. During the final incubation period, the inoculated host cells were removed from the incubator and used with Optimem^®The medium was washed twice. At 10min, 1.6ml of Optimem^®Adding to Lipofectin^®The/carrier mixture and 1ml of the resulting mixture was added to both cell wells. The host cells were again placed in the incubator and allowed to stand for 5 hours, and at 5 hours, Lipofectin was removed^®The/carrier mixture was replaced with standard cell growth medium.

From 18 to 24 hours after medium change, from 50 to 100. mu.l of cell growth medium is removed from the tissue culture plate and the sample is placed in AUSZYME^®Reaction pool in Monoc Ional diagnostic kit (Abbott laboratories, Abbott Park, IL)And analyzing the antigen expression condition. The volume of the sample to be tested was adjusted to 200. mu.l with PBS, and then 50. mu.l of conjugate and reaction microspheres were added to each sample. The reaction cell was incubated at 40 ℃ for 80min, after which all liquid reaction components in the wells were washed away. The reaction microspheres were transferred to a new tube, after which 300. mu.l of the chromogenic reaction buffer was added. After 30 minutes, the color reaction was terminated by adding 1M sulfuric acid, and the absorbance of the reaction was measured at 490 nm. The data shown in figure 1 is the average absorbance of both wells in two experiments.

As shown in FIG. 1, the introduction of RIA, HBV 3' UTR or both into the basic vector (CMV promoter, exon and polyadenylation region) increased the expression of HBsAg in SCC15 cells. As shown in fig. 2, introduction of chicken keratin or chicken heart actin intron into the basic vector (CMV promoter, exon, HBV 3' UTR and polyadenylation region) increased expression of HBsAg in SCC15 cells.

Example 10 in vitro analysis of beta-gal vector series antigen expression

SSC-15 or B16 host cells are transfected as described in example 9.

At 18 to 40 hours after medium exchange, medium supernatant was removed and cells were washed with PBS. After removal of the wash solution, 500. mu.l lysis buffer (50mM NaPO) was used₄0.1% Triton X-100, pH7) for 5min to lyse the cells, which were then physically scraped off the plastic dish. The lysate was centrifuged for two minutes to remove cell debris, and 10 to 25. mu.l of the clarified lysate were added to 500. mu.l of reaction buffer (80ug/ml o-nitrophenylgalactosylceramide), 50mM NaPO₄pH7) and incubated at 37 ℃ for 10 to 20 minutes. Add 500. mu.l of 1M Na₂CO₃The reaction was stopped and the absorbance read at 405 nm. Data are expressed as the ratio of expression of the enhancement vector (containing introns, HBVenh, or both) to expression of the base vector.

Addition of RIA, HBV 3' UTR or both to the basic vector (CMV promoter, exon and polyadenylation region) increased the expression of beta-gal in both cell lines. FIG. 3 shows the results obtained with SCC15 cells. The addition of chicken keratin or chicken heart actin intron to the basic vector (CMV promoter, exon, HBV 3' UTR and polyadenylation region) increased the expression of β -gal in both cell lines. Fig. 2 shows the results obtained with B16 cells.

Example 11 in vitro analysis of the expression of HSV gD vector series antigens

SSC15 or B16 host cells were transfected as described in example 9. 18 hours after transfection, plates were ice-washed for 15 min. The wells were then washed with 2ml PBS (Biowhittaker, Walkwille, Md.). Cells were fixed with 0.05% PBS diluted glutaraldehyde (Polysciences Inc, Warrington, PA) and incubated at room temperature for 30 min. All subsequent incubations were continued at room temperature for 1 hour and between each incubation, washing was performed as described above. Cells in the plate were blocked with 2ml of 5% milk powder (in PBS) (Bio Rad Laboratories, Melville, NY). Followed by 2% milk powder/PBS/0.05% Tween-20^®(Sigma, St. Louis, MO) 1: 1000 dilution of anti-gD monoclonal antibody (ABI, Columbia, MD)1ml and PBS/0.1% Tween-201: 2500 dilution of goat anti-mouse HRP (KPL, Gaithersburg, MD)1ml incubation. The chromogenic reaction was performed with 1ml TMB microwell substrate (BioFX, Owings Mills, Md.). 1M H ₂SO₄The reaction was terminated, the liquid was transferred to a plastic cuvette and the optical density value was read at 450 nm. Data are expressed as the ratio of expression of the enhancement vector (containing introns, HBVenh, or both) to expression of the base vector.

Introduction of RIA with or without HBV 3' UTR into the basic vector (CMV promoter, exon and polyadenylation region) increased expression of HSV gD in both cells. Fig. 4 shows the results obtained by SC 15.

Example 12 in vitro analysis of the expression of the SEAP vector series of antigens

SSC15 or B16 host cells were transfected as described in example 9. The medium supernatant was removed 18 to 40 hours after the medium change, and the supernatant was heated at 70 ℃ for 30 min. 10-25. mu.l of the heat-inactivated supernatant was incubated with 1/10 volumes of 100mM I-homoarginine for 5 min. Mu.l of alkaline phosphatase reaction buffer (cat #172-1063, Bio-Rad, prepared according to the instructions) was added to the lysate and incubated at 37 ℃ for 10 to 20 min. The reaction was stopped by adding 500. mu.l of 1M NaOH and the absorbance read at 405 nm. Data are expressed as the ratio of expression of the enhancer vector (containing introns, HBVenh, or both) to expression of the base vector, or the ratio of expression of the experimental set signal peptide to expression of the human TPA signal peptide vector.

As shown in fig. 5, introduction of RIA, HBV 3' UTR or both into the basic vector (CMV promoter, exon and polyadenylation region) increased SEAP expression in B16 cells. Unexpectedly, the mere introduction of the HBV 3' UTR into the basic vector (CMV promoter, exon and polyadenylation region) increased SEAP expression in SCC15 cells.

The addition of the signal peptide of bovine aprotinin, chicken lysozyme or tobacco extensin to the N-terminus of mature SEAP provides the possibility for efficient secretion of SEAP into the cell culture supernatants of both cell lines. Fig. 6 shows the results for B16 cells.

Example 13 in vitro analysis of antigen expression of the human IgG Fc fragment Signal peptide series

SSC15 or B16 host cells were transfected as described in example 9. The medium supernatant was removed 18 to 40 hours after medium exchange.

ELISA plates (Costar) were loaded with 100. mu.l goat anti-human IgG (Sigma # I3382, diluted with carbonate coating buffer 1/1000) per well and incubated overnight at 4 ℃. All subsequent incubations were continued at room temperature for 1 hour and washed with washing solution (10mM Tris, 150mM NaCl, 0.1% Brij-35, pH8.0) between each incubation. Then blocked with 100. mu.l of 5% milk powder (in PBS) followed by dilution with dilution buffer (2% milk powder, PBS, 0.05% Tween-20) ^®) Serial dilutions of the culture supernatants were incubated together. Then combined with 100. mu.l of goat anti-human IgG/HRP per well (Sigma # A6029, diluted in dilution buffer 1/5000)Incubate and then perform a color reaction with 100. mu.l TMB microwell substrate. Using 100. mu.l of 1M H₂SO₄The reaction was stopped and the absorbance read at 405 nm. Data are presented as the ratio of expression of the experimental set of signal peptides to the expression of the human TPA signal peptide vector.

The signal peptide of bovine aprotinin, chicken lysozyme or tobacco extensin is added to the N-terminal of the human Fc fragment, which provides possibility for the effective secretion of hFc into cell culture supernatants of two cell lines. Fig. 6 shows the results for B16 cells.

Example 14 immunization of mice with HBsAg, HSVgD and Flu-M2 plasmid expression vectors Use of

(a) Preparation of immunization cartridges

For each plasmid to be tested, 25mg of 2 micron gold powder was weighed into a centrifuge tube. After addition of a 250. mu.l aliquot of 50mM spermidine (Aldrich Chemical, Inc, Milwaukee, Wis.), the tubes were shaken and sonicated for a short period of time. The gold powder was removed by centrifugation and replaced with 100. mu.l of fresh spermidine. The gold powder was resuspended by shaking, and then 25. mu.g of DNA was added to the tube and mixed. While gently shaking the tube, 100. mu.l of 10% CaCl was added ₂(Fujisawa USA, Inc, Deerfield, IL) to precipitate DNA on gold beads. The precipitation reaction can be carried out on a bench top for 10min, followed by collecting the gold beads by short centrifugation and washing three times with absolute ethanol (Spectrum Quality Products, Inc, Gardena, CA) to remove excess precipitation reagent. The washed gold bead/DNA complex was then resuspended in 3.6ml of 0.05mg/ml polyvinylpyrrolidone (360KD, Spectrum Quality Products, Inc, Gardena, Calif.) in absolute ethanol. This slurry was then injected into Tefzel tubes (McMaster-Carr, Chicago, IL) located in tube spinners (PowderJectVaccines) and the inside of the Tefzel tubes was coated with the gold bead/DNA complex. After the coil process was completed, the tubes were cut into 0.5 "vaccine" boluses "that were loaded into an XR1 device (PowderJect Vaccines) for delivery to mice.

(b) Inoculation procedure

A mixture of Ketaset (Fort Dodge) and Rompun (Bayer) was used to anaesthetise mice of 4 to 6 weeks of age. The abdomen was shaved with an electric hair cutter to remove body hair, and two non-overlapping "shots" of vaccine were delivered to the shaved area by an XR1 device (450 psi). Animals were returned to cages and blood was collected 6 weeks after inoculation. Balb/c mice were used to evaluate the HBsAg expression vector, Swiss Webster mice were used to evaluate the HSV-gD and Flu M2 expression vectors.

Analysis of anti-HBsAg antibodies in serum

Blood samples were taken from the vaccinated animals on the sixth week. Sera isolated from these blood samples were placed into wells of a reaction cell of an AUSAB ® EIA diagnostic kit (Abbott Laboratories, Abbott Park, IL). The volume of serum added depends on the antibody titer of the sample, which is diluted with sample dilution buffer to fall within the values obtainable with the quantitative analysis chart. 200 μ l of each vial from an AUSAB ® quantitative analysis plate (Abbott laboratories, Abbott Park, IL) was added to the well of the reaction cell. One microsphere was added to each well, the reaction cell was then sealed and incubated at 40 ℃ for two hours, and then all the liquid reaction components in the wells were washed away. 200 μ l of the conjugate mixture was added to each washed well, followed by sealing the reaction cell and incubating at 40 ℃ for two hours, and then washing off all liquid reaction components in the wells. The beads were transferred to a new tube and 300. mu.l of the chromogenic reaction buffer was added. The color reaction was stopped by adding 1M sulfuric acid at 30 minutes and the absorbance of the reaction was measured using a Quantum II ® spectrophotometer (Abbott Laboratories, Abbott Park, IL) at 490 nm. The spectrophotometer can calculate the antibody level of the sample by comparing the absorbance of the sample to a standard curve generated from a quantitative map. Antibody levels were then corrected with dilution factor. The data shown in FIG. 7 are the geometric mean titers of all animals vaccinated with a particular vector.

Analysis of anti-Flu M2 antigen antibody in serum

Synthetic Flu M2 peptide (QCB/Biosource, Hopkinton) was used at a concentration of 1. mu.g/mlMA 96-well Costar medium was coated onto a Costar medium binding ELISA plate (Fisher Scientific, Pittsburgh, Pa.) in PBS (Biowhittaker, Walkerville, Md.) and incubated overnight at 4 ℃. Plates were washed three times with 10mM Tris (Sigma, St. Louis, MO)/150mM NaCl (Fisher Scientific)/0.1% Brij-35(Sigma) and then blocked with 5% milk powder (Bio Rad Laboratories, Melville, N.Y.) (in PBS) for 1 hour at room temperature. All subsequent incubations were performed at room temperature for 1 hour and washed between each incubation as described above. Sample mouse serum, standards (high titer, anti-M2 mouse serum) and negative controls (anti-HBsAg mouse serum) were diluted 2% milk powder/PBS/0.05% Tween-20(Sigma) and incubated in ELISA plates. Using 2% milk powder/PBS/0.05% Tween-20^®Goat anti-mouse IgG (H + L) biotin-conjugated antibody (Southern Biotechnology Association, Birmingham, AL) was diluted 1: 8000 followed by dilution of streptavidin-horseradish peroxidase conjugate (Southern Biotechnology) with PBS/0.1% Tween-201: 8000. The chromogenic reaction was performed with the substrate TMB (BioFX, Owings Mills, Md.). With 1M H ₂SO₄The reaction was stopped and absorbance read at 450nm with a microplate reader (Molecular Devices, Sunnyvale, Calif.). Endpoint titers were calculated using SoftMaxPro 4.1 software (Molecular Devices), four parameter analysis, and the titers were normalized against standard sera with known titers to minimize variation between groups and within groups. The results are shown in FIG. 7.

Analysis of anti-HSV gD antigen antibodies in serum

96-well Costar medium binding ELISA plates (Fisher Scientific, Pittsburgh, Pa.) were coated with HSV gD (Viral Therapeutics, Ithaca, NY) protein (dissolved in PBS (Biowhittaker, Walkerville, Md)) at a concentration of 1. mu.g/ml and incubated overnight at 4 ℃. Plates were washed three times with 10mM Tris (Sigma, St. Louis, MO)/150mM NaCl (Fisher Scientific)/0.1% Brij-35(Sigma) and then blocked with 5% milk powder (Bio Rad Laboratories, Melville, N.Y.) (in PBS) for 1 hour at room temperature. All subsequent incubations were performed at room temperature for 1 hour and washed between each incubation as described above. 2% milk powder/PBS/0.05% Tween-20(Sigma) dilutionsSerum from this mouse, standard (high titer, anti-gD mouse serum) and negative control (anti-HBsAg mouse serum) and incubated in ELISA plates. Goat anti-mouse IgG (H + L) biotin-conjugated antibody (Southern Biotechnology Association, Birmingham, AL) was diluted with 2% milk powder/PBS/0.05% Tween-201: 8000 followed by dilution of streptavidin-horseradish peroxidase conjugate (Southern Biotechnology) with PBS/0.1% Tween-201: 8000. The chromogenic reaction was performed with the substrate TMB (BioFX, Owings Mills, Md.). With 1M H ₂SO₄The reaction was stopped and absorbance read at 450nm with a microplate reader (Molecular Devices, Sunnyvale, Calif.). Endpoint titers were calculated using SoftMaxPro 4.1 software (Molecular Devices), four parameter analysis, and the titers were normalized against standard sera with known titers to minimize variation between groups and within groups. The results are shown in FIG. 7.

Example 15 construction of plasmid ppJV1671, influenza NDA vaccine vector

Plasmid pppjv 1671 was constructed which encodes and expresses Hemagglutinin (HA) antigen of influenza a/panama/2007/99(H3N 2).

The construction of pPJV1671 is best described in three main steps:

(i) cloning an expression gene;

(ii) reforming a carrier framework; and are

(iii) And (5) transforming a final plasmid.

Cloning of expressed genes

The coding sequence for influenza virus HA was obtained by standard reverse transcriptase polymerase chain reaction (RT-PCR) cloning techniques using the A/panama/2007/99 virus obtained from the disease control and prevention center (Atlanta, GA) as a source of template ribonucleic acid (RNA).

The following steps were applied to clone the expressed gene HA:

● dsDNA fragments were generated by RT-PCR from RNA segment 4 of A/Panama/2007/99(H3N 2);

● amplification of RNA segment 4 DNA clones in a vector based on standard pUC19 in E.coli;

● sequencing the H3 Panama HA coding sequence in the RNA segment 4 clone; and are

● A second PCR reaction was performed to form a DNA fragment containing the H3 Panama HA coding sequence (without the ATG codon) that was terminally compatible with the pPJV7563 DNA vaccine "empty" vectors (NheI and Bsp 120I).

The vector framework pPJV7563 is transformed

The plasmid backbone of ppJV1671 is pPJV 7563. Most of the plasmid backbone sequence in pppJV 7563 is also present in pWRG7128, a DNA vaccine vector that has been evaluated in several human clinical trials. This section briefly describes the construction of pppjv 7563. A detailed flow chart is provided in FIG. 14 for the construction of pPJV7563 and pPJV1671, and the following table comparison of key elements in plasmid pWRG7128 and pPJV1671 was made (Table 2). A map of pPJV1671 is shown in FIG. 16.

TABLE 2

Comparison of key elements in the vector backbone:

plasmid WRG7128(HBsAg) and pPJV1671 (influenza virus HA)

Description of the changes	Reasons or purposes for the change	Altered biological effects or meanings or possible effects
Description of the changes	Reasons or purposes for the change	Altered biological effects or meanings or possible effects	The kanamycin resistance gene fragment in pppJV 1671 is 354 bases shorter than that used in pWRG 7128.	In order to remove foreign sequences from the vector	Without negative changes to predictable vaccine efficacy or safety
The Sph1-Pst1 linker upstream of the CMV promoter in pWRG7128 was removed.	In order to remove foreign sequences from the vector. Making these restriction sites more efficient for the construction of potential vaccines.	Without negative changes to predictable vaccine efficacy or safety		In order to remove foreign sequences from the vector
			Intron a of CMV was removed and CMV exon 1 had been fused to the remaining 9 bases of CMV exon 2 in pppjv 1671.	The CMV exons were fused to reform the 5' UTR of the spliced pWRG7128 transcript.	Fusion of CMV exons does not alter vaccine efficacy or safety profiles.
Intron a of the rat insulin gene was inserted into the 5' -UTR of the fused CMV exon and HbsAg.	To replace the CMV intron a sequence with a functional sequence.	The addition of rat insulin intron a to the vaccine vector has been shown to enhance antigen expression and subsequent antibody response.
			The polyadenylation region was changed from bovine growth hormone to rabbit β -globin.	In order to use another polyadenylation signal.	Without negative changes to predictable vaccine efficacy or safety

The final plasmid pPJV1671 is transformed

There are two major steps involved in engineering the final plasmid pPJV1671 from the backbone pjv7563 (as shown in fig. 14), which are:

● deletion of the Nhe1-Bgl II site from pPJV 7563; and are

● the H3Panama HA coding sequence was inserted into pPJV7563 to generate the final pPJV1671, H3Panama HA DNA vaccine vector.

Complete sequence analysis of pPJV1671 confirmed the complete vector backbone and HA coding sequence as listed in table 3 below.

Comparison of pWRG7128 and pPJV1671

The major differences in vector backbone between clinically examined pWRG7128 encoding HBsAg and the influenza vaccine pPJV1671 vector encoding H3Panama HA are shown in table 2 above. These differences include the replacement of the human cytomegalovirus (hCMV) intron a element with rat insulin intron a, and the replacement of the bovine growth hormone polyadenylation sequence with the rabbit β -globin polyadenylation sequence. A large number of animal studies with the pPJV1671 showed good influenza virus DNA vaccine performance in both small and large animals. Plasmid pppJV 1671 also has good performance in humans after PMED of this DNA vaccine as described in example 16 below.

Characteristic and functional map of pPJV1671

A functional map of pPJV1671 is shown in FIG. 16. The characteristics of the map are shown in Table 3. Plasmid pppjv 1671 uses the hCMV immediate early promoter and the 5' non-coding sequences of exons 1 and 2. The promoter was linked to the rat insulin intron A and the 5' UTR of the HBV pre-S2 gene. Translation of the H3Panama HA coding sequence begins at the ATG codon, which is fixed in the vector in the context of the consensus Kozak translation initiation sequence. The HA coding sequence is followed by the transcriptional enhancer of HBV (HBV enh). Finally, the rabbit β -globin polyadenylation site contributes to transcription termination.

Since the natural ATG translational start codon of the HA gene of A/Panama/2007/99(H3N2) does not coincide with the Kozak translational start consensus sequence, the ATG element provided by the pPJV7563 DNA vaccine vector was chosen to initiate translation. Antigen expression of the DNA vaccine vector was enhanced using ATG codons identical to Kozak consensus sequence. The use of the ATG codon provided by the vector (by insertion at Nhe I site) resulted in a very small two amino acid insertion at the amino-terminus of the coding sequence of the HA gene (as shown in figure 16).

Source of nucleotide sequences in pPJV1671

The history of the detailed construction and sequencing of pppjv 1671 allowed the localization of the source of all nucleotide positions within the plasmid. BLAST alignments were performed between vector component sequences and GenBank databases to confirm that the correct component positioning had indeed been performed.

TABLE 3

Identification of the Components contained in plasmid pPJV1671

Base numbering	Component identification
Base numbering	Component identification	1-896	Kanamycin resistance gene flanked by Tn903 sequences (Tn 9)03. pUC4K residues 1-44 and 861-896)
897-902	Sal 1 cloning site/pUC 19MCS	1-896
897-902	Sal 1 cloning site/pUC 19MCS	903-1587	CMV promoters
1588-1718	CMV exon 1/2 fusion	903-1587	CMV promoters
1588-1718	CMV exon 1/2 fusion	1719-1724	BamH1/Bgl 2 fusion
1725-1857	Rat insulin intron A	1719-1724	BamH1/Bgl 2 fusion
1725-1857	Rat insulin intron A	1858-1863	BamH1 cloning site
1864-1984	Non-coding preS2 region of HbsAg	1858-1863	BamH1 cloning site
1864-1984	Non-coding preS2 region of HbsAg	1985-1987	ATG initiation codon
1988-1993	Nhe1 cloning site	1985-1987	ATG initiation codon
1988-1993	Nhe1 cloning site	1994-3688	H3N2HA coding sequence
3689-3691	Stop codon	1994-3688	H3N2HA coding sequence
3689-3691	Stop codon	3692	G nucleotides of H3Panama 3' UTR
3693-3698	Bsp 120I cloning site	3692	G nucleotides of H3Panama 3' UTR
3693-3698	Bsp 120I cloning site	3699-4231	HBV enhancer
4232-4242	Cloning of the artificial sequence. Unknown origin	3699-4231	HBV enhancer
4232-4242	Cloning of the artificial sequence. Unknown origin	4243-4373	Rabbit beta-globin polyadenylation region
4374-4379	EcoR1 cloning site	4243-4373	Rabbit beta-globin polyadenylation region
4374-4379	EcoR1 cloning site	4380-5446	PUC19 vector

pPJV1671 sequence (Master cell Bank)

Qiagen Megaprep DNA prepared from the newly constructed pPJV1671 plasmid was used for sequencing by PowderJect Research Department. The observed sequence data of pppJV 1671 is 100% identical to its theoretical sequence. Sequencing during the preparation process also showed that the plasmid sequence also matched 100% of the theoretical and studied sequences. The individual plasmid elements and their positions in the sequence are shown in table 3 above.

Example 16 non-clinical evaluation of pPJV1671Estimation: monovalent influenza virus DNA in large animal models Immunogenicity of vaccine pPJV1671

The immunogenicity of pppjv 1671 was studied using a pig model, the formation of which is described in example 15 above. Eight weeks old domestic white pigs (growing-finishing pigs) were used in this study. Hemagglutination Inhibition (HI) antibody titers were measured in candidate study animals, and all study animals had hemagglutination inhibition antibody titers of < 1: 10.

Two groups of animals (8 per group) were immunized by PMED with pPJV1671DNA vaccine. Both groups were primed and boosted with a 4 week interval. Each immunization consisted of two sequential (side-by-side) administrations to the skin. The dose of DNA vaccine per administration was 1. mu.g of DNA concentrated to the surface of 0.5mg of gold particles. DNA vaccine delivery was carried out using an XR-1 clinical and research set-up operating at 500psi helium pressure.

Pigs were immunized on day 0, boosted on day 28, and bled two weeks after boost on day 42. Sera were tested for HI antibody response using standard HI assay. The results of this study are shown in fig. 17, which shows that the pppjv 1671DNA vaccine caused significant HI antibody titers in 100% of the tested animals, the mean HI antibody titer was much greater than the alternative HI antibody titers used against human influenza virus, which were 1: 40.

Example 17 clinical trials evaluating influenza virus DNA vaccine constructs in humans

Introduction to the design reside in

A phase I escalating dose clinical study was conducted to evaluate the safety and immunogenicity of the pppjv 1671DNA vaccine, a monovalent PMED influenza virus DNA vaccine containing the HA (hemagglutinin) gene from a/Panama/2007/99(H3N 2). Safety was assessed by monitoring local and systemic adverse outcomes from immunization to study completion. Immunogenicity was assessed by measuring serum Hemagglutination Inhibition (HI) antibody levels.

Three groups of healthy adult subjects (12 per group) were given a single dose of DNA vaccine (1, 2 or 4 μ g) on day 0 delivered at 1, 2 or 4PMED dosing. At all 3 dose levels, PMED influenza DNA vaccines elicited serum Hemagglutination Inhibition (HI) antibody responses, and the highest and most consistent level of response was elicited in subjects vaccinated with the highest dose level. Antibody responses were most significant at day 56, the last time point tested. Treatment-related reactions occur limited to mild to moderate skin reactions at the site of inoculation, and these skin reactions are mostly self-limiting. These results provide an indication of the safety and immunogenicity of the influenza virus DNA vaccines formed.

Materials and methods

Vaccine and delivery system

For clinical plasmids, the HA coding sequence was obtained by standard reverse transcriptase-polymerase chain reaction (RT-PCR) cloning techniques using samples of the A/Panama/2007/99 virus as a source of template RNA, a gift to J.Katz for disease control and prevention centers. This H3 Panama HA coding sequence was inserted into the pPJV7563 vector as described in example 15 above, thereby obtaining the final pPJV1671H3 Panama HA DNA vaccine vector. Complete sequence analysis of pPJV1671 confirmed the entire vector backbone and HA coding sequence.

Plasmid pppjv 1671 was prepared in Strathman GmbH (Hannover, Germany) with good preparative practice. The Vaccine was used according to the method described previously (Roy et al, (2001)19: 764-78) plasmid DNA was coated onto 1-3 μm gold particles and used to prepare PMEDs using a PowderJect XR-1 device. Each dose contained a nominal value of 1. mu.g of DNA coated on 0.5mg of gold. Vaccine quality parameters analyzed included the amount of gold and DNA, expression in vitro, immunocompetence in mice, and absence of bioburden and endotoxin.

Patient population and environment

The clinical phase of the study was performed at the MDS pharmacy Services (MDS Pharma Services, Lincoln, NE) and was agreed to by the local institutional review board. Thirty-six (36) adult volunteers were enrolled (table 4).

Table 4: demographic profile of study population

	Group 1	Group 2	Group 3
	Group 1	Group 2	Group 3	The average age (years) age range (years) for each group of men versus women before immunization HAI GMT (range)	123120-487∶516(5-40)	123120-504∶817(5-40)	123221-495∶712(5-40)

Subjects were considered appropriate if they met the following conditions: healthy, between 19 and 50 years of age, not pregnant or lactating, can provide written test volunteers, and have pre-immunization Hemagglutination Inhibition (HI) titers of ≥ 10 and ≤ 40 for influenza virus A/Panama.

Reasons for exclusion included immunosuppressive treatment in the last 6 months, a history of skin disease, scarring, nevi, incisions or tattoos at the site of immunization, allergic response to gold, a history of morganard treatment, influenza vaccine in the last 12 months, flu-like symptoms or confirmed diagnosis of influenza in the current season, or any medical condition in which influenza immunization is not recommended.

All subjects, except for two, completed the study and were evaluated for safety and immunogenicity. One subject failed follow-up and the other did not cooperate. However, since all 36 subjects provided a sample before immunization, received immunization, and were evaluated at least once for safety and immunogenicity after immunization, evaluation data of safety and immunogenicity of all subjects were obtained. All the protocols followed the international conference drafted in 1975, the declaration of helsinki by the Edinburgh revision (2000) in Scotland, and the harmonized guidelines for good clinical practice (step 4, May 1996).

Design of clinical study

Subjects were divided into three treatment groups in order, 12 people each. Each PMED DNA vaccine administration delivered 0.5mg of 1. mu.g DNA on gold, plasmid pPJV1671(H3 Panama). A single immunization dose was given to the medial upper arm of the first group on day 0. Subjects in group 2 were immunized twice with a total of 2 μ g of DNA vaccine administered to adjacent sites inside the upper arm on day 0. Subjects in group 3 were dosed 4 times on day 4 for a total of 4 μ g DNA. Administration of the Vaccine using a PowderJect XR-1 device with 500psi helium pressure has been shown to be well tolerated in previous studies (Roy et al (2001) Supra, Rottinghaus et al (2003) Vaccine21(31): 4604-8 and Tacket et al (1999) Vaccines17(22)：2826-9)。

Clinical safety and immunogenicity assessment

Safety can be assessed by monitoring vaccination site reactions during the study and recording the incidence of local and systemic adverse outcomes. All subjects were evaluated for vaccine safety and local tolerance at the time of immunization, 1 hour and 2 hours after immunization. Subjects were again evaluated for local tolerance on days 3, 7, 14, 21, 28, 56, and 180. Systemic adverse outcomes were monitored by physical examination, vital signs, laboratory safety tests, and anti-double stranded DNA antibody tests. These tests were performed before and after immunization.

The immunogenicity of each vaccine can be determined using a modification of the previously described methods (Kendal, et al (1982) concentrates and products for laboratory-based in-fluenza surveillance. US Department of Health and human Services, Public Health Service, Centers for Disease Control: B17-35), by collecting blood samples on day 0 (pre-immunization) and on days 14, 21 and 56 post-immunization, and determining the HAI titer against A/Panama/2007/99.

Clinical data analysis

Safety data are listed in the table. Each HI titer is expressed as the Geometric Mean (GMT) of two independent tests, which typically gave identical or similar results. If the titer of the sample is less than the assay limit of 10, it is assigned to titer 5. For each group, the titer of log transformation can be used to calculate the GMT at each time point and the 95% Confidence Interval (CI). For each time point, the GMT ratios of the respective dose groups relative to the reference value were compared by chi-square test using the procedure CATMOD in SAS. Differences between GMTs in three groups were also compared.

Seroconversion (percentage of subjects with HAI titres increased by a factor of 4 or more with respect to the titre before immunization) and seroprotection (percentage of subjects with titres of 40 or more obtained after immunization) were calculated and compared for each group by chi-square test using the program CATMOD in SAS, which compares the percentage of responder cells by using log units as a function of the relation of the dependent variable.

Results

Safety and reaction occurrence

Local responses were evaluated for a total of 84 vaccine administration sites (fig. 18). The local skin response scores for all three groups (84 vaccination sites in total) were averaged. Skin reactions were scored according to the following criteria:

-erythema: 0-none; 1 ═ reddening; 2, light black red; 3 ═ beet red.

-edema: 0-none; 1-slightly thickened; 2-significant thickening; large firm hive (3).

-color change: 0-none; 1 ═ slightly visible; 2-a significant change in color; 3-brown shoe polish-like.

-exfoliation: 0-none; 1 ═ e.g. fine white dandruff; peeling off the sun burn sample; thick yellow scab 3.

-itching/discomfort: 0-none; 1 ═ slight itching or slight tenderness; 2-moderate itching or tenderness.

As expected based on previous studies (Roy et al (2001), Rottinghaus et al (2003) and pocket et al (1999), all supra), subjects experienced a local skin reaction, but no local reaction in the severe (score 3) range occurred. No bleeding or skin damage was recorded. The typical local response is characterized by mild to moderate erythema, edema and skin color changes, with occasional itching/discomfort followed by mild superficial skin exfoliation.

Rare local reactions include petechiae (2/84 site), minor contusions (2/84 site) and minor scabs (15/84 site). Although edema resolved 14 days after inoculation, erythema and skin color changes persisted for 28 days. Of the 84 total vaccination sites, 30 remained with a slight change in skin color at day 56 post-vaccination, 21 at day 180. One subject also had detectable contusion at 2 sites on day 180.

Systemic adverse effects observed during the study were mild and considered treatment-independent. The results reported from day 7 to 56 after immunization included headache (11 in 10 subjects), fatigue (4 in 3 subjects), myalgia (3 in 1 subject), fever (2 in 2 subjects), cold hand sensation (2 in 2 subjects), back pain (2 in 1 subject) and vomiting, joint pain, muscle tone, shivering, intermittent hypertension and intermittent hypotension (1 in each case). No antibodies against double stranded DNA were detected. The overall local and systemic adverse outcome status of all study groups provided an indication of the safety of influenza DNA vaccines delivered by PMED.

Antibody response

Subjects were pre-screened to select subjects with HI titers of > 10 and < 40 against influenza A/Panama, and some subjects had titers of < 10 on the day of vaccination. The baseline HI titers were all < 40, and there was no significant difference in the baseline titers between groups (p ═ 0.439, see table 4 above).

Immunization resulted in a significant increase in Geometric Mean Titer (GMT) at all time points in all three groups (as shown in table 5 below).

Table 5:

serum antibody response, seroconversion and seroprotection

Group by group	Sky	Seroconversion^a(％)	Serum protection^b(％)	Mean GMT rise (fold)
Group by group	Sky	Seroconversion^a(％)	Serum protection^b(％)	Mean GMT rise (fold)	1	0142156	-8(1/12)17(2/12)33(4/12)	17(2/12)42(5/12)33(4/12)58(7/12)	-1.41.72.8^c
2	0142156	-17(2/12)8(1/12)67(8/12)	33(4/12)50(6/12)58(7/12)92(11/12)	-1.72.13.9	1	0142156	-8(1/12)17(2/12)33(4/12)	17(2/12)42(5/12)33(4/12)58(7/12)	-1.41.72.8^c
2	0142156	-17(2/12)8(1/12)67(8/12)	33(4/12)50(6/12)58(7/12)92(11/12)	-1.72.13.9	3-	0142156	-17(2/12)33(4/12)64(7/11)	8(1/12)25(3/12)67(8/12)100(11/11)	-1.83.48.1

Seroconversion is defined as the change from a negative titer (. ltoreq.10) before inoculation to a titer. gtoreq.40 after inoculation, or a significant increase in the antibody titer, i.e.when the titer before inoculation is. gtoreq.10, there is at least a four-fold increase between the titer before inoculation and the titer after inoculation

b seroprotection Rate is defined as the proportion of subjects who received a titre of 40 or more after immunization

c the values meeting the CPMP criteria are in bold

Although there was a tendency for HI titers and seroconversion to rise in a dose-response manner, there was no statistical difference in GMT, seroprotection or seroconversion between these three groups. For all three vaccine dose levels, GMT, seroconversion and seroprotection rates increased with increasing time post-vaccination, with the highest titers appearing at day 56 (see table 5). In the first group, 33% of subjects experienced seroconversion by day 56, 58% achieved seroprotective HAI titers, with a 2.8-fold increase in GMP. In the second group, the seroconversion at day 56 was 67%, 92% of subjects were seroprotected and had a 3.9-fold increase in GMT. The highest and most consistent titers were observed in the third group at day 56, when 100% seroprotection was observed, with an 8.1-fold increase in GMT relative to baseline. In this group, seroconversion was less than 100% because some of the seroprotected subjects did not show a titer rise of > 4-fold due to the relatively high baseline HAI titer.

Discussion of the related Art

The work described in this example provides the first successful development of an anti-influenza antibody response that is expected to be effective in preventing influenza.

Immunological analysis indicated that all dose levels produced an anti-influenza antibody response on average. Comparisons made against the CPMP (proprietary medical commission) guidelines for approved annual influenza vaccines showed that the 1 μ g dose group achieved the standard for Geometric Mean Titer (GMT) on day 56, the 2 μ g dose group achieved the requirements for all three standards (seroconversion, seroprotection and GMT) on day 56, while the 4 μ g dose group achieved the standard for GMT on day 21 and the requirements for all three standards on day 56.

A total of 320 treatment-induced adverse outcomes (AEs), including vaccine site reactions, were reported in 34 of 36 subjects administered (94%). 1 SAE, a foot bone injury, reported during the study was considered unlikely to be relevant to the study vaccine. Seven (7) subjects experienced AEs that should be reported to the FDA that were all considered unlikely to be relevant to the study vaccine. Most (71%) AE levels were lighter. The most common AE reported was headache (53% of all subjects). No subject discontinued the study due to AE. A total of 89 vaccination-site-associated AEs were reported in 27 out of the 36 subjects dosed, with 12 subjects (33%) reporting mild dosing-site pain as the most common local AE. A measure of the absence of local reactions was assessed as tertiary (severe) and was therefore considered an adverse outcome. Typical vaccination site reactions include redness/erythema, edema, scaling, color change and scab/scar formation.

In summary, administration of pppjv 1671 was well received in all subjects and elicited an effective anti-influenza antibody response. The highest dose of 4 μ g meets the criteria set by CPMP approved annual influenza vaccines on day 21.

Example 18 non-clinical evaluation of trivalent DNA vaccines in pigs

Three DNA vaccine vectors encoding HA molecules of the three 2001-2002 influenza vaccine strains were constructed to evaluate the immunogenicity of candidate human trivalent influenza DNA vaccines in relevant animal models. Since human and pig skin are very similar in structure, in the past, domestic pigs were used as a model for human PMED DNA vaccines. The relevance of the pig model as a predictive model of human PMED DNA vaccine performance has been demonstrated in phase I human clinical trials of hepatitis b surface antigen DNA vaccines, where good vaccine performance in pigs is reflected in humans.

Samples of three 2001-one 2002 human influenza vaccine strains (A/Panama/2007/99(H3N2), A/NewCaledonia/20/99(H1N1), and B/Victoria/5/00) were obtained from the disease control center (CDC) and used in reverse transcriptase/polymerase chain reaction (RT-PCR) experiments to form DNA fragments encoding the corresponding HA antigens. The following steps were used to develop the final three HA DNA vaccine vectors:

● dsDNA fragments were generated by RT-PCR from RNA segment #4 of A/Panama/2007/99(H3N2), A/New Caledonia/20/99(H1N1) and B/Victoria/5/00.

● in E.coli, RNA segment #4DNA clones were amplified in a vector based on standard pUC 19.

● HA coding sequences were sequenced in RNA segment #4 clone.

● A second series of PCR reactions (based on authentic HA gene sequence data) was performed to form DNA fragments from each virus containing the HA coding sequence (without ATG codons) whose ends were compatible with pPJV7563DNA vaccine expression vectors (Nhe I and Bsp 120I).

● three fragments of the HA coding sequence were inserted into the clinical DNA vaccine vector pPJV7563, thus generating the final three DNA vaccine vectors.

● sequence analysis of the HA coding sequence in all three vectors confirmed the absence of mutations.

The resulting vector uses the chimeric promoter of the present invention. Thus, the vector uses the human cytomegalovirus (hCMV) immediate early promoter and 5' non-coding sequences from immediate early exons 1 and 2. The promoter is linked to rat insulin intron A and the 5' untranslated region (UTR) of the Hepatitis B Virus (HBV) pre-S2 gene. Translation of the HA coding sequence begins at the ATG codon, which is fixed in the vector in the context of a consensus Kozak translation initiation sequence. The HA coding sequence is followed by the transcriptional enhancer of HBV (HBV enh). Finally, the rabbit β -globin polyadenylation site contributes to transcription termination.

Since the native ATG translation initiation codon of the HA gene does not coincide with the Kozak translation initiation consensus sequence, the ATG element provided by the pPJV7563DNA vaccine vector was used to initiate translation. Antigen expression of DNA vaccine vectors is generally improved using ATG codons consistent with the Kozak consensus sequence. The use of the ATG codon provided by the vector (by insertion at Nhe I site) results in a very small two amino acid insertion at the amino terminus of the coding sequence for the HA antigen.

The immunogenicity of these vectors in trivalent formulations was evaluated in a pig model. The three carriers were mixed 1: 1 and prepared on the surface of gold particles at a ratio of 2. mu.g total DNA/mg gold, using 0.5mg gold per administration. Each immunization consisted of two subsequent PMED administrations, totaling 2. mu.g DNA and 1mg gold. The vaccination protocol included two such immunizations spaced four weeks apart. Pigs that did not suffer from influenza were divided into two immunization groups based on the delivery device actually used. Trivalent influenza DNA immunization was performed on a group of animals using a reusable "research" PMED device that has been successfully used to elicit immune responses against multiple antigens in multiple animals. A second group of animals was immunized using a clinical device used to successfully vaccinate humans with HBV DNA vaccine. The latter device differs from the research device in that it uses a disposable "one shot" plastic nozzle instead of a reusable 12-shot nozzle connected to the research device.

Two weeks after the second immunization, serum samples were collected and blood coagulation inhibition (HI) antibody titers specific for homologous H3, H1, and B viruses were measured. These data are shown in FIG. 19. 100% seroconversion to H3 and B antigens was observed using both devices, with the geometric mean HI titers greatly exceeding the 1: 40 titer replacement level that is normally considered necessary for immunization against influenza virus. The data obtained suggest that the technology platform will be able to elicit significant responses in humans.

H1-specific immune responses were low. This was later determined to be due to rearrangement of the H1HA gene that occurred in the H1DNA vaccine vector during plasmid preparation. This rearrangement is not detected in the first analysis of DNA prior to preparation and inoculation, but is detected in a more stringent analysis. Based on the results using H3 and B vectors, the H1-specific response would be significantly enhanced if a functional plasmid was utilized.

Example 19 construction of plasmid pppjv 2012

Plasmid pppjv 2012 was constructed. pppjv 2012 expresses the a and B subunits of the thermolabile toxin (LT) of enterotoxigenic e. Expression of a functional LT toxin by pppjv 2012 in vivo can be used to enhance immune responses against other proteins expressed from plasmids co-administered with pjv 2012.

LT is an 84kD multi-subunit protein comprising a pentamer of an a subunit and the same B subunit. LT is expressed and secreted by e.coli and binds to GM1 ganglioside on enterocytes through pentamers of the B subunit. After toxin uptake, the A subunit activates to produce excess cAMP and disrupts electrolyte balance inside and outside the intestinal lumen (Tauschek, et al (2002) Proc Natl Acad Sci, USA99: 7066-7071). Thus, for the biological activity of LT expressed in vivo, there is a need to produce a and B subunits and signal sequences that mediate secretion from expressing cells. Plasmid vectors encoding the A and B subunits of LT have been shown to enhance the immune response elicited against several viral antigens when delivered together using specific mediated epidermal delivery (Arrington et al (2002) J Virol 76 (9): 4536-46).

In addition to the coding sequences for the a and B subunits of the LT toxin, pppjv 2012 contains the chimeric promoter sequence of the present invention, a rabbit β globin poly a sequence, a signal sequence that mediates secretion from the expressing cells, a kanamycin resistance gene, and a bacterial origin of replication that ensures efficient gene expression. This plasmid is shown in FIG. 22.

Plasmid pppjv 2012 was constructed by the following steps:

● LTA coding sequences were amplified from E.coli genomic DNA by PCR and inserted into an intermediate plasmid;

● the LT A coding sequence is excised and inserted into an "Acceptor" plasmid under the control of the CMV promoter.

● the LT B coding sequence was amplified from E.coli genomic DNA by PCR and inserted into an intermediate plasmid under the control of a truncated CMV promoter.

● excise the LT A expression cassette.

The resulting plasmid pppjv 2012 expresses both subunits of LT in mammalian cells.

promoter and enhancer sequences in ppPJV 2012

The LT A subunit is expressed from the CMV Immediate Early (IE) promoter. The LT B subunit is expressed from a truncated CMV IE promoter. For both genes, additional sequences are included to enhance expression, particularly HBVpre-S25' UTR (Moriary et al (1981) Proc Natl Acad Sci USA 78: 2606-. To ensure secretion from expressing cells, the chicken lysozyme signal peptide (CLSP, Genbank accession CR390743) has been inserted into both LT subunits. In addition, the HBVenv enhancer (Vannice and Levinson (1988) J Virol.62: 1305-.

Cloning of LT A and B subunits

Coli strain E078: h11 was obtained from ATCC (accession No. 35401) and the a and B subunits were amplified by separate PCR reactions using primers homologous to the 5 'and 3' ends, designed with reference to the GenBank accession number AB011677 file. The coding sequences for the two subunits formed do not include sequences encoding bacterial signal peptides present at the amino terminus. The a and B subunit fragments were ligated separately into plasmid WRG 7054.

Construction of pPJV2012

The LT A coding sequence was excised and ligated to the vector backbone of pPJV7592 and a fragment derived from pPJV7572 comprising the CMV promoter, the 5' untranslated region and the CLSP. The resulting plasmid was designated a 1. Cutting A1 and connecting with the fragment from pPJV7592 to reform a multiple cloning site, and the obtained plasmid is called Acceptor; this provides the LT a component of the pPJV 2012.

The LT B coding sequence was excised and ligated to the vector backbone of pPJV7591 and the fragment derived from pjv7572 containing the 3' end of the untranslated region and CLSP. The resulting plasmid was designated Donor, thus providing the LT B component of pPJV 2012.

To form pPJV2012, the fragment from the Donor, containing the LT B expression cassette, was ligated with the vector fragment from Acceptor. The resulting plasmids express both LT A and LT B in mammalian cells.

Source of nucleotide sequence in pppJV 2012

pppjv 2012 had been fully sequenced and aligned with the database to specify the source of each sequence. The resulting sequence was identical to that expected (shown in table 6 below).

Table 6:

identification of the Components contained in plasmid pPJV2012

Base numbering	Component identification
Base numbering	Component identification	1-905	Sequences derived from pUC4K including kanamycin resistance gene
906-1038	Rabbit beta globin poly A	1-905
906-1038	Rabbit beta globin poly A	1039-1351	LT B subunit coding sequence
1352-1357	Linker sequences	1039-1351	LT B subunit coding sequence
1352-1357	Linker sequences	1358-1417	Chicken lysozyme signal sequence
1418-1538	5' UTR of HBV pre-S2	1358-1417	Chicken lysozyme signal sequence
1418-1538	5' UTR of HBV pre-S2	1539-1544	Linker sequences
1545-1677	Rat intron A	1539-1544	Linker sequences
1545-1677	Rat intron A	1678-1683	Linker sequences
1684-1814	CMV exon 1/2	1678-1683	Linker sequences
1684-1814	CMV exon 1/2	1815-1935	Truncated CMV IE promoter
1936-1947	Linker sequences	1815-1935	Truncated CMV IE promoter
1936-1947	Linker sequences	1948-2632	CMV promoters
2633-2763	CMV exon 1/2	1948-2632	CMV promoters
2633-2763	CMV exon 1/2	2764-2769	Linker sequences
2770-2902	Rat insulin intron A	2764-2769	Linker sequences
2770-2902	Rat insulin intron A	2903-2908	Linker sequences
2909-3029	5' UTR of HBV pre-S2	2903-2908	Linker sequences
2909-3029	5' UTR of HBV pre-S2	3030-3089	Chicken lysozyme signal peptide
3090-3095	Linker sequences	3030-3089	Chicken lysozyme signal peptide
3090-3095	Linker sequences	3096-3818	LT A coding sequence
3819-3830	Linker sequences	3096-3818	LT A coding sequence
3819-3830	Linker sequences	3831-4363	HBV env enhancer sequence
4364-4374	Unknown sequence	3831-4363	HBV env enhancer sequence
4364-4374	Unknown sequence	4375-4050	Rabbit beta globin poly A
4051-5578	Sequences derived from pUC19	4375-4050	Rabbit beta globin poly A

In addition, BLAST searches were conducted for sequence homology to the entire human genome, looking for sequence similarity between sequence segments as short as 19 bases. "no significant similarity found"; the sequence has the highest homology with the 68 base rGpA sequence, but the longest section of the same sequence is only 18 bases.

pPV2012 comparison of sequences with pPJV1671

The sequence of pPJV2012 was compared to the sequence of pPJV 1671. The results are shown in Table 7 below.

Table 7: comparing sequences of pPJV2012 and pPJV1671

Base numbering of sequences in pPJV2012	Sequence is also present in pPJV1671 influenza virus DNA vaccine?
Base numbering of sequences in pPJV2012		1-896	Is that
897-905	Whether or not	1-896	Is that
897-905	Whether or not	906-1038	Is that
1039-1351	Whether or not	906-1038	Is that
1039-1351	Whether or not	1352-1357	Is that
1358-1417	Whether or not	1352-1357	Is that
1358-1417	Whether or not	1418-1538	Is that
1539-1544	Is that	1418-1538	Is that
1539-1544	Is that	1545-1677	Is that
1678-1683	Is that	1545-1677	Is that
1678-1683	Is that	1684-1814	Is that
1815-1935	Is that	1684-1814	Is that
1815-1935	Is that	1936-1947	Is that
1948-2632	Is that	1936-1947	Is that
1948-2632	Is that	2633-2763	Is that
2764-2769	Is that	2633-2763	Is that
2764-2769	Is that	2770-2902	Is that
2903-2908	Is that	2770-2902	Is that
2903-2908	Is that	2909-3029	Is that
3030-3089	Whether or not	2909-3029	Is that
3030-3089	Whether or not	3090-3095	Is that
3096-3818	Whether or not	3090-3095	Is that
3096-3818	Whether or not	3819-3824	Is that
3825-3830	Is that	3819-3824	Is that
3825-3830	Is that	3831-4363	Is that
4364-4374	Is that	3831-4363	Is that
4364-4374	Is that	4375-4505	Is that
4512-5578	Is that	4375-4505	Is that

Analysis indicated that 4418 base pairs (79%) of 5578 base pairs in pppjv 2012 were also present in pPJV1671 influenza virus DNA vaccine. In addition to the coding sequences for LT a and B subunits, 4418 base pairs (97%) of the 4544 base pairs in pppjv 2012 were also present in pjv 1671.

Note that biodistribution/integration studies were also performed on pPJV1671, and no evidence of integration was found in each case.

Example 20 plasmid pPJV 7788-a plasmid for expression of the e.coli LT subunit in mammalian cells Construction of the plasmid

Plasmid pPJV7788 was formed using the following plasmids:

ppJV7563 and pPJV7592, derived from pPJV7275, pPJV7284, pPJV7389, pPJV7586 and pPJV 7293.

pppJV 7590, a derivative of ppJV7389, contains a Multiple Cloning Site (MCS) immediately upstream of the CMV promoter.

pppjv 7572, a derivative of pPJV7389, contains the coding sequence of the Chicken Lysozyme Signal Peptide (CLSP) immediately upstream of the encoded antigen. This allows secretion of the c-terminally fused antigen outside the cell.

(i)Construction of pPJV7785(LTA Acceptor plasmid)

Sal 1 cleaves pPJV7563, blunts the ends, and cleaves with Nde1 to form a vector fragment. Sph1 cleaves pPJV7590, blunted and cleaved with Nde1 to form an insert containing the 5' end of the MCS and CMV promoters. These fragments were ligated to form pppjv 7592.

Pppjv 7592 was cut with Nco1 and Bgl2 to form a vector fragment. pPJV7572 was cut with Nco1 and Nhe1 to form an insert containing the CMV promoter 3 'end, 5' untranslated region and CLSP. pPJV2004 was cut with Nhe1 and BamH1 to form an insert containing LTA subunits. These fragments were ligated to form plasmid pppjv 7785.

(ii)Construction of pPJV7787(LTB Donor plasmid)

Pppjv 7389 was cut with Nco1 and EcoR1 to form a vector fragment. Pppjv 7572 was cut with Nco11 and Nhe1 to form an insert containing the 3 'end of the CMV promoter, the 5' untranslated region, and CLSP. pppJV 2005 was cut with Nhe1 and BamH1 to form an insert containing LTB subunits. pPJV7586 was cut with Bgl2 and EcoR1 to form an insert containing the rabbit β -globin polyadenylation region. These fragments were ligated to form pppjv 7787.

(iii)Construction of pPJV7788

ppPJV 7785 was cut with Xho1 and Mfe1 to form a vector fragment containing the LTA expression cassette. pPJV7787 was cut with Xho1 and EcoR1 to form an insert containing the LTB expression cassette. These fragments are ligated to form pppJV 7788, which expresses LTA and LTB subunits when introduced into mammalian cells.

Table 8 below provides the location of the various elements in construct pPJV 7788.

Table 8: component ID of pPJV7788

Base numbering	Components
Base numbering	Components	1-4445-860861-98398410011002-16861687-18171818-18231824-19561857-19621963-20832084-21432144-21492150-24622463-25932594-26232624-33083309-34393440-34453446-35783579-3584	Tn903, pUC4K residual KanR (Tn903) Tn903, pUC4K residual pUC19MCSCMV ProCMV exon 1/2Bam/Bgl fusion rat insulin intron ABamH1 site HBV pre-S2 5' -UTR lysozyme SPNhe1 site LTBRBGpA multiple cloning site CMV ProCMV exon 1/2Bam/Bgl fusion rat insulin intron ABamH1

3585-37053706-37653766-36713772-44944495-45064507-50395040-50505051-51815182-51875188-6254

5' -UTR lysozyme SPNhe1 site LTA polymeric linker HBVenh unknown origin rGlob pAEcoR1 site pUC19 of HBV pre-S2

Example 21-pPML 7789-expression of H5/VN 1194A hemagglutinin protein in mammalian cells Construction of seed plasmid-

Another construct pPML7789 was formed which was capable of expressing the H5/VN1194 hemagglutinin protein using the chimeric promoter of the invention. The construct pPJV7563 described in example 5 was used as backbone for the construct pml 7789.

Plasmids containing the coding sequence of the H5/VN1194 HA gene were used as templates for PCR. PCR primers were designed at the 5 'and 3' end sites to insert pPJV 7563. The ppJV7563 was cut with Nhe1 and Bsp120I to form vector fragments. The PCR fragment amplified from the H5/VN1194 template was cut with the same enzyme to form an insert. These two fragments were ligated to produce pPML 7789. The coding sequence and flanking sequences were verified by sequencing.

The nucleotide positions of the individual elements in pml7789 are shown in table 9 below.

Table 9: components of construct pPML7789

Nucleotide position		Component
Nucleotide position		Component	Starting point: 1	End point: 44	Tn903, pUC4K residue
Starting point: 45	End point: 860	KanR(Tn903)	Starting point: 1	End point: 44	Tn903, pUC4K residue
Starting point: 45	End point: 860	KanR(Tn903)	Starting point: 861	End point: 896	Tn903, pUC4K residue
Starting point: 897	End point: 902	pUC19MCS	Starting point: 861	End point: 896	Tn903, pUC4K residue
Starting point: 897	End point: 902	pUC19MCS	Starting point: 903	End point: 1587	CMV Pro
Starting point: 1588	End point: 1718	CMV exon 1/2	Starting point: 903	End point: 1587	CMV Pro
Starting point: 1588	End point: 1718	CMV exon 1/2	Starting point: 1719	End point: 1724	Bam/Bgl fusions
Starting point: 1725	End point: 1857	Rat insulin intron A	Starting point: 1719	End point: 1724	Bam/Bgl fusions
Starting point: 1725	End point: 1857	Rat insulin intron A	Starting point: 1858	End point: 1863	BamH1 site
Starting point: 1864	End point: 1984	5' -UTR of HBV pre-S2	Starting point: 1858	End point: 1863	BamH1 site
Starting point: 1864	End point: 1984	5' -UTR of HBV pre-S2	Starting point: 1985	End point: 1993	ATG-Nhe
Starting point: 1994	End point: 3699	VN1194H5	Starting point: 1985	End point: 1993	ATG-Nhe
Starting point: 1994	End point: 3699	VN1194H5	Starting point: 3700	End point: 3705	Bsp120I site
Starting point: 3706	End point: 4238	HBVenh	Starting point: 3700	End point: 3705	Bsp120I site
Starting point: 3706	End point: 4238	HBVenh	Starting point: 4239	End point: 4249	Unknown linker sequence
Starting point: 4250	End point: 4380	rGlob pA	Starting point: 4239	End point: 4249	Unknown linker sequence
Starting point: 4250	End point: 4380	rGlob pA	Starting point: 4341	End point: 4341	PolyA sites
Starting point: 4381	End point: 4386	EcoR1 site	Starting point: 4341	End point: 4341	PolyA sites
Starting point: 4381	End point: 4386	EcoR1 site	Starting point: 4387	End point: 5453	pUC19

Example 22 immunization with DNA Using HSV-2ICP27 and E.coli heat labile toxin Effective protective cellular immune response is formed

Materials and methods

Virus

HSV-2 virus (MS strain, ATCC VR-540 and HG52 strain G, a gift from Nancy Sawtell at the university of Sincinagi) was cultured on VERO cells (ATCC CCL-81) and purified in a sucrose gradient prior to use.

DNA vaccines and DEI vectors

A plasmid encoding HSV-2ICP27 (FIG. 27) was formed by inserting a PCR fragment containing the entire ICP27 gene (UL54) into pTarget (Promega, Madison, Wis.). PCR fragments were amplified from purified genomic DNA of the HSV-2MS strain using the 5 '(GCCACTCTCTTCCGACAC, SEQ ID NO: 63) and 3' (CAAGAACATCACACGGAAC, SEQ ID NO: 64) primers. The amplified sequence corresponded to nucleotides 114,523 to 116,179 of the published genomic sequence of HG52 clone of HSV-2 (GenBank accession No. NC _ 007198). The entire pICP27 was sequenced by the DNA sequencing core facility at university of Wisconsin, Madison.

Plasmid PJV2012 encodes the a and B subunits of LT of a single vector and is shown in fig. 22. The construction of pppjv 2012 is described in example 19. In pppjv 2012, the LT a subunit gene is expressed by a transcription unit consisting of: human cytomegalovirus (hCMV) immediate early promoter, hCMV (non-coding) fused exons 1 and 2, rat insulin intron a, the 5 'non-coding region of the Hepatitis B Virus (HBV) pre-S2 gene, the ATG codon, the lysozyme signal peptide coding sequence, the LT a subunit coding sequence, the 3' non-coding HBV transcriptional enhancer region, and the rabbit β -globin polyadenylation sequence.

The pppjv 2012 also encodes the LT B subunit product of another transcription unit that extends in the opposite direction (fig. 22). The LT B transcriptional unit is similar to the transcriptional unit encoding the LT a product, but does not contain additional copies of hCMV and HBV transcriptional enhancer elements. hCMV and HBV enhancer elements located in LT a transcription units are also useful for LT B transcription units.

Plasmid PJV2013 (not shown) is similar to pPJV2012, but encodes the a and B subunit products of cholera toxin from a single vector. The functional map of pPJV2013 is the same as that shown in pjv2012 in fig. 22.

DNA vaccination by particle-mediated epidermal delivery (PMED)

To 6 to8 week old Balb/c mice were inoculated with PMED DNA as described previously (Arrington et al, J.Virol).764536-4546, 2002), except that each immunization consisted of one PMED delivery to the sub-abdominal skin, with each delivery containing 0.5mg gold coated with a total of 0.5 μ g of dna vaccine/DEI vector formulation. Two such "single-shot" immunizations were performed 4 weeks apart, and animals were sacrificed or challenged 2 weeks after the second or "booster" immunization.

Peptide pool and peptides

The ICP27 amino acid sequence derived from MS strain virus was used as a library template. The ICP27 protein between HG52 and the MS sequence (see FIG. 28 and SEQ ID NO: 65) is highly conserved, therefore, the library was able to detect responses to both strains. A peptide library (Mimotopes, Fisher Scientific) encompassing the entire sequence of ICP27 was synthesized using peptides 18 amino acids long, 11 amino acids of which overlap with adjacent peptides. A total of 72 peptides were prepared.

To help identify positive peptides, methods were used with Tobery etalj25459-66, 2001 divide the library into peptide pools. Peptide pools were formed using the method shown in table 10. There were 12 peptide pools (named C1-C12, 6 peptides per pool) and 6 peptide pools (R1-R6, 12 peptides per pool). Peptide pools containing peptides #45 and #46 are shown in bold.

Table 10: peptide pool composition

	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10
	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	R1	1	3	5	7	9	11	13	15	17	19
R2	25	27	29	31	33	35	37	39	41	43	R1	1	3	5	7	9	11	13	15	17	19
R2	25	27	29	31	33	35	37	39	41	43	R3	49	51	53	55	57	59	61	63	65	67
R4	4	6	8	10	12	14	16	18	20	22	R3	49	51	53	55	57	59	61	63	65	67
R4	4	6	8	10	12	14	16	18	20	22	R5	28	30	32	34	36	38	40	42	44	46
R6	52	54	56	58	60	62	64	66	68	70	R5	28	30	32	34	36	38	40	42	44	46

	C11	C12
	C11	C12	R1	21	23
R2	45	47	R1	21	23
R2	45	47	R3	69	71
R4	24	26	R3	69	71
R4	24	26	R5	48	50
R6	72	2	R5	48	50

ELISPOT assay

As previously described, IFN- γ ELISPOT assays were performed on fresh spleen and lymph node cells, except that the antigen used for stimulation was a single peptide or pool of peptides as defined in the results portion of each experiment. In some cases, prior to ELISPOT analysis, the T cell population was removed by magnetic beads (Dynal) according to the manufacturer's instructions.

Micro-sample multi-index flow protein quantification (CBA) analysis

Two weeks after the second immunization, fresh splenocytes were collected and plated in SM (RPMI medium + 10% fetal bovine serum supplemented with MEM sodium pyruvate, non-essential amino acids and 2-mercaptoethanol (Invitrogen, Carlsbad, Calif.)) at 1X 10⁷Cell/ml resuspension. Cell samples were processed at 1X 10⁶Cells (100. mu.l) per well were seeded in 96-well flat-bottom plates. Splenocytes were treated with 100 μ l aliquots of ICP27 peptide dissolved in SM or in the medium itself. Final peptide concentration was 10^-8And M. Control splenocytes from untreated animals were inoculated with and without peptide and with and without Con-A (final concentration of 2.5mg/ml) and used as negative and positive controls, respectively.

Plates were incubated at 37 ℃ for 48 hours, then 160 μ l of each sample was collected and stored at-20 ℃ until analysis using the BD Cytometric Bead Array (CBA) mouse Th1/Th2 kit (BDBiosciences, San Jose, CA (cat. # 551287)). Briefly, the kit uses 5 populations of microspheres with different fluorescence intensities coated with capture antibodies specific for mouse IL-2, IL-4, IL-5, INF-gamma and TNF-alpha. These five microsphere populations were mixed together to form an array resolved on the FL3 channel of a BD FACSCalibur flow cytometer. Cytokine capture microspheres are mixed with PE-conjugated detection antibodies and then incubated with standards or test samples to form sandwich complexes. The results were obtained after sample collection using BD CBA analysis software. The intensity of the sample for each cytokine was compared to a standard curve to quantify the cytokine concentration. The analysis was carried out on pure samples or samples diluted 1: 10 according to the manufacturer's instructions.

HSV-2 virus challenge

Using 30. mu.l of a solution containing about 50LD₅₀(2×10⁶PFU) HSV strain MS PBS an intranasal challenge was performed on anesthetized Balb/c mice. Mice were observed for 20 days post infection and scored for morbidity and mortality. Incidence rates were scored according to the following list on a scale of 0 to 4: 4, health; 3, the fur shakes and sneezes; 2, soreness of the eyes or buttocks, decreased mobility; 1, bowing back, almost inactive; 0, death. Mice treated with IFN- γ - (eBioscience, San Diego, CA (cat. #16-7311)) and/or TNF- α - (cat. #16-7332) specific monoclonal antibodies before and after challenge were injected intraperitoneally with injections containing 90 μ g of antibody on days-2, 0, 2, 4, 6, and 8 from the time of virus challenge. In the T cell elimination experiments, injections containing 200. mu.g of an antibody specific to CD4 or CD8 cell population (Functional Grade purified anti-mouse CD4, eBioscience Cat #: 16-0041; Functional Grade purified anti-mouse CD8, eBioscience; Cat #: 16-0081) were intraperitoneally injected on days-2 and 0 from the virus challenge.

Results

Identification of a Strong CD8 epitope in Balb/c mice

To identify T cell responses to ICP27 protein, IFN- γ ELISPOT analysis was performed on cells obtained from Balb/C and C57B1/6 mice infected with a virulent strain of HSV-2 (HG52 strain G). A peptide library containing peptides encompassing the entire ICP27 coding sequence was used to stimulate T cells. The peptide is 18 amino acids long and overlaps with the adjacent peptide by 11 amino acids. As described in the materials and methods section and table 10, the peptide pool contained 6 peptides (peptide pool C1-C12) or 12 peptides (peptide pool R1-R6), an arrangement that helped identify positive peptides in the peptide pool. Splenocytes and lymph node cells obtained from Balb/C mice 7 days post infection showed very strong responses to peptides in peptide pools C10, C11, R2 and R5 (FIG. 29A), while weak responses to peptides in several other peptide pools. Only a weak response was detected in cells obtained from C57B1/6 mice. It was found that the pattern of positive responses was similar in the spleen and lymph node cell populations analyzed.

Based on the IFN-. gamma.ELISPOT results, two adjacent peptides (#45, #46) were presumed to contain dominant T cell epitopes. This was confirmed by testing each peptide separately (data not shown). Peptides 45 and 46 stimulated strong IFN-. gamma.secretion and contained the homologous nine amino acid sequence HGPSLYRTF (FIG. 29B, SEQ ID NO: 68) which was predicted to bind to the Dd allele. Notably, peptide 46 also contains a region corresponding to the epitope of ICP27 from HSV-1 in the Balb/c mice previously described (Banks et al, j.virol.67,613-616, 1993). Comparison of the ICP27 sequences for HSV-1 and HSV-2 shows that this region differs by one amino acid: the alanine (A) residue in HSV-2ICP27(LYRTFAANPRA, SEQ ID NO: 69) was replaced by glycine (G) in HSV-1(LYRTFAGNPRA, SEQ ID NO: 70). However, even if this peptide elicits a very strong response, this region is not present in peptide 45.

To determine the relative importance of the two possible epitopes of peptide 46 in the IFN-. gamma.ELISPOT assay, peptides LYRTFAANPRA and HGPSLYRTF were synthesized and tested. Splenocytes isolated from infected mice responded strongly to HGPSLYRTF peptide, but no response above background to LYRTFAANPRA sequence was detected. A strong CD8+ ICP27 response was obtained using magnetic beads to remove the T cell population, followed by IFN- γ ELISPOT analysis. These data indicate that a strong CD8 response was developed in Balb/c mice specifically against the sequence HGPSLYRTF of the HSV-2ICP27 protein.

In vitro immune response to ICP27 vaccine

The ICP27 sequence of the MS strain of HSV-2 was used to form the DNA vaccine "plcp 27" (figure 27). The construct was found to differ from the disclosed HG52ICP27 gene sequence by only two nucleotides as determined (fig. 28). A change of nucleotide G at position 57 to a (silent change) and a change of a to C at position 484 would change the lysine in the HG52 strain to asparagine in the MS form. Therefore, the DNA vaccine expressed MS strain ICP27 protein was almost identical to the HG52 strain form of the protein, and there was no difference in the putative CD8 epitope region.

pICP27DNA vaccine was used to immunize Balb/C and C57B1/6 mice with PMED. Splenocytes and lymph node cells were tested against the peptide pool plot described in FIG. 29 in an IFN- γ ELISPOT assay. The positive response pattern against ICP27 peptide observed in DNA-immunized mice was the same as that observed in infected mice (data not shown).

In vivo response to ICP27 vaccine

The in vivo activity of the immune response resulting from pICP27DNA vaccination was studied in a Balb/c mouse model of prophylactic intranasal infection. Infection with different doses of HSV-2MS strains in 12-week-old Balb/c mice confirms LD ₅₀About 3 × 10⁴PFU (data not shown). In DNA protection experiments, 50LD was used₅₀The virus challenge dose of (a), since this dose always causes 100% mortality in non-immunized animals.

A series of experiments showed that immunization with pICP27 DNA alone (prime and boost) had limited ability to protect mice. Thus, to improve protection, DEI vectors encoding Cholera Toxin (CT) or escherichia coli heat Labile Toxin (LT) were administered with ICP27DNA vaccines. For each of these two toxins, both the a and B toxin subunits are expressed on the same plasmid.

Mice were immunized with ICP27 vaccine alone or supplemented with CT or LT DEI vector at a ratio of 9: 1 (0.45. mu.g antigen DNA + 0.05. mu.g DEI vector DNA). The immunisation group used consisted of 16 animals per group, half of which were challenged with virus and the other half of which were sacrificed on challenge with ICP27 specific cytokine product.

The results of this study are shown in figure 31. The results show that immunization with ICP27+ CT and ICP27 formulation alone can provide partial protection and no protection, respectively, whereas 100% protection was observed when using the ICP27+ LT formulation (fig. 31A). These results demonstrate that co-delivery of the DEI vector with the ICP27 vector can enhance protection and can potentiate the superiority of LT over CT as an immunostimulant in DNA immunization. The quantification of the cytokines produced by the CD8 epitope peptide after in vitro stimulation using the CBA kit showed a good correlation between challenge survival and the levels of INF- γ (fig. 31B) and TNF- α production (fig. 31C).

Effect of cytokines and T cell population

The role of ICP 27-specific IFN-. gamma.or TNF-. alpha.production in protecting challenged animals was evaluated. Animals vaccinated with pICP27+ pPJV2012(LT) were treated for several days with INF-gamma and/or TNF-alpha specific monoclonal antibodies before and after challenge to neutralize these cytokines in vivo. The incidence data are shown in figure 32. As before, animals immunized with ICP27+ LT DEI were completely protected from challenge and exhibited only a mild short-term morbidity (1 out of 4 animals were shaky in fur), while 100% of naive mice or mice immunized with pICP27 vector alone died from challenge. In the group immunized with the ICP27+ LT DEI formulation and subsequently treated with anti-TNF-alpha, one example of death due to anesthetic complications was observed immediately after the intranasal challenge injection. Importantly, the remaining three mice showed only brief skin-hair trembling and were fully recovered, suggesting that TNF- α production may not contribute significantly to protection. In contrast, two groups of mice immunized with ICP27+ LT DEI receiving anti-IFN- γ or anti-IFN- γ + anti-TNF- α at challenge became extremely unhealthy with 7 out of 8 animals dying, clearly indicating the importance of IFN- γ as an essential regulator of protection.

To test the protective effect of the CD4 and CD8 populations, these cell populations were removed using CD4 or CD8 antibodies and then subjected to infection challenge (fig. 33). A meaningful pattern appeared 20 days after follow-up observation of the mortality of the mice. As expected, mice given plcp 27 with empty vector were not significantly protected from infection. pICP27+ LT DEI vaccination provided 100% survival, but removal of CD4 and CD8 cells from similarly immunized mice abolished the protective effect of the vaccination and resulted in sensitivity levels in the mice that were identical to those of non-immunized mice. In mice immunized with the pICP27+ LT DEI vaccine, infection after removal of the CD8 cell population caused the mice to die of infection, suggesting that these cells play an important role in protecting against infection or encephalitis. Removal of the CD4 cell population in mice vaccinated with plcp 27+ LT DEI caused a nine day delay (relative to non-immunized mice) before morbidity and mortality in these mice, suggesting that CD4T cells had an effect on long-term survival.

Example 23: immunogenicity of influenza virus H5N1 DNA vaccine in mice

Materials and methods

DNA plasmid pPML7789 (example 21, fig. 20) encoding HA of influenza a/Vietnam/1194/2004[ H5N1] virus was deposited on the surface of gold particles and delivered to mice by particle-mediated epidermal delivery (PMED) using the proprietary delivery technique of PowderMed. Delivery can be performed with or without another plasmid pppjv 2012 encoding the escherichia coli heat labile toxin subunits a and B (example 19, fig. 22) as an adjuvant. This other plasmid was deposited on the surface of the same gold particles together with the pPML7789 plasmid in the presence of the pPJV 2012. This can be achieved by: the pppjv 2012 and pml7789 were first premixed in liquid form in a weight ratio of 1: 9, and then these plasmids were co-deposited on the surface of a population of gold particles. As a negative control, gold particles without deposited plasmid were delivered by PMED. The mice were as follows:

Strain: Balb/C mice

Number: 36

Sex: female

Body weight range: 15.0-19.6g (on the first day of operation)

Age: day 43-49 (arrive on the day)

Diet: RM1 particles

The supplier: charles River UK Ltd

The mice were divided into six groups as shown in table 11 below. The manipulations performed on the mice and the number of days the manipulations were performed are shown in table 12 below. HAI analysis was performed on sera according to standard methods.

Table 11: animal treatment ingredient distribution

Group number #	Mode of administration	Number of animals per group	Handling of conditions
Group number #	Mode of administration	Number of animals per group	Handling of conditions	1	Epidermis	6	Negative control 1 dose day-1 blood draw, day 14 blood draw and sacrifice
2	Epidermis	6	Negative control 2 doses were collected on days-1 and 21, collected on day 36 and sacrificed	1	Epidermis	6
2	Epidermis	6		3	Epidermis	6	pPML77891 doses were bled on day-1, bled on day 14 and sacrificed
4	Epidermis	6	pPML77892 doses were collected on days-1 and 21, collected on day 36 and sacrificed	3	Epidermis	6
4	Epidermis	6		5	Epidermis	6	pPML7789+ pPJV20121 doses collected on day-1, day 14 and sacrificed
6	Epidermis	6	pPML7789+ pPJV20122 doses collected on days-1 and 21, day 36 and sacrificed	5	Epidermis	6

Table 12: the following operations were performed on the days marked X

Days of study	0	14	21	35
Days of study	0	14	21	35	Transponder transplantation	X
Body weight	X	X	X^b	X^b	Transponder transplantation	X
Body weight	X	X	X^b	X^b	Inoculation of	X		X^b
Health score	X	X	X^b	X^b	Inoculation of	X		X^b
Health score	X	X	X^b	X^b	Antibody serum	X	X^a	X^b	X^b
Collecting spleen		X^a		X^b	Antibody serum	X	X^a	X^b	X^b
Collecting spleen		X^a		X^b	Sacrifice of		X^a		X^b

^a1, 3 only&5 groups of

^b2 nd and 4 th only&6 groups of

Results

The assay was performed on NIBRG-14[ H5N1]HAI titer of Virus

All controls have been observed to be within acceptable limits. The negative control (NIBRG-14[ H5N1] virus) was negative on all plates. Positive controls (turkey red blood cells) were positive on all plates. The second positive control (serum containing anti-influenza A/Chicken/Scotland/59[ H5N1] virus antibodies) gave a geometric mean of 28, 40, 56 or 80HAIU (within +/-2 fold of the acceptable limit of 40 HAIU) on all plates.

Except for day 21 ID: 1074 (group 6, this sample could not be collected) and day 21 ID: 1059 (group 4, this sample did not obtain sufficient serum to perform the assay), all samples on days 0, 14 and 21 were < 10HAIU (below the limit of detection).

For day 35 blood sampling, except for ID: 1074 (group 6) in addition to the inability to draw blood, the geometric mean titer of each sample was calculated, along with the group arithmetic mean, median and standard deviation for each group. These results are shown in Table 13 below.

Table 13: 2, 4 th&HAI titer of sera obtained on day 35 of group 6

Group by group	Sample numbering	Geometric mean titer	Arithmetic mean	Median value	SD
Group by group	Sample numbering	Geometric mean titer	Arithmetic mean	Median value	SD	2	104710481049105010511052	＜10＜10＜10＜10＜10＜10	＜10	＜10	0
4	105910601061106210631064	284040202020	28	24	10	2	104710481049105010511052	＜10＜10＜10＜10＜10＜10	＜10	＜10	0
4	105910601061106210631064	284040202020	28	24	10	6	107110721073107410751076	160226160^*160113	164	160	40

Failure to obtain data due to premature death of one animal

These results indicate that the pml7789 plasmid is immunogenic in mice, and that this immunogenicity is enhanced by the addition of pppjv 2012.

Thus, novel nucleic acid constructs, compositions comprising the various constructs, and nucleic acid immunization techniques using the constructs are described. While the preferred embodiments of the present invention have been described in detail, it should be understood that: modifications may be made without departing from the spirit and scope of the invention.

Sequence listing

<110> Baodejicket vaccine GmbH (POWDERJECT VACCINES, INC)

<120> nucleic acid construct

<130>N93730C GCW

<150>UK 0507997.5

<151>2005-04-20

<150>US 60/672,479

<151>2005-04-19

<150>US 60/648,382

<151>2005-02-01

<160>70

<170>PatentIn version 3.1

<210>1

<211>685

<212>DNA

<213> human cytomegalovirus

<400>1

aatattggct attggccatt gcatacgttg tatctatatc ataatatgta catttatatt 60

ggctcatgtc caatatgacc gccatgttga cattgattat tgactagtta ttaatagtaa 120

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 180

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 240

tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 300

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt 360

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac 420

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 480

tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 540

cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 600

cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 660

ataagcagag ctcgtttagt gaacc 685

<210>2

<211>131

<212>DNA

<213> human cytomegalovirus

<400>2

gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc 60

gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt gccaagagtg 120

actcaccgtc c 131

<210>3

<211>135

<212>DNA

<213> rat (Rattus ratus)

<400>3

atcagcaagc aggtatgtac tctccagggt gggcctggct tccccagtca agactccagg 60

gatttgaggg acgctgtggg ctcttctctt acatgtacct tttgctagcc tcaaccctga 120

ctatcttcca ggtca 135

<210>4

<211>955

<212>DNA

<213> Artificial sequence

<220>

<223> chimeric promoter sequence

<400>4

aatattggct attggccatt gcatacgttg tatctatatc ataatatgta catttatatt 60

ggctcatgtc caatatgacc gccatgttga cattgattat tgactagtta ttaatagtaa 120

tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 180

gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 240

tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 300

cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt 360

gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac 420

tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 480

tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 540

cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt 600

cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat 660

ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt 720

gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga 780

acgcggattc cccgtgccaa gagtgactca ccgtccggat ctcagcaagc aggtatgtac 840

tctccagggt gggcctggct tccccagtca agactccagg gatttgaggg acgctgtggg 900

ctcttctctt acatgtacct tttgcttgcc tcaaccctga ctatcttcca ggtca 955

<210>5

<211>121

<212>DNA

<213> hepatitis B virus

<400>5

cagagtcagg ggtctgtatt ttcctgctgg tggctccagt tcaggaacag taaaccctgc 60

tccgaatatt gcctctcaca tctcgtcaat ctccgcgagg actggggacc ctgtgacgaa 120

c 121

<210>6

<211>57

<212>DNA

<213> herpes simplex virus

<400>6

ataagctgca ttgcgaacca ctagtcgccg tttttcgtgt gcatcgcgta tcacggc 57

<210>7

<211>48

<212>DNA

<213> hepatitis B virus

<400>7

ctttgtacta ggaggctgta ggcataaatt ggtctgttca ccagcacc 48

<210>8

<211>533

<212>DNA

<213> hepatitis B virus

<400>8

taacaaaaca aaaagatggg gttattccct aaacttcatg ggttacgtaa ttggaagttg 60

ggggacattg ccacaagatc atattgtaca aaagatcaaa cactgtttta gaaaacttcc 120

tgtaaacagg cctattgatt ggaaagtatg tcaaaggatt gtgggtcttt tgggctttgc 180

tgctccattt acacaatgtg gatatcctgc cttaatgcct ttgtatgcat gtatacaagc 240

taaacaggct ttcactttct cgccaactta caaggccttt ctaagtaaac agtacatgaa 300

cctttacccc gttgctcggc aacggcctgg tctgtgccaa gtgtttgctg acgcaacccc 360

cactggctgg ggcttggcca taggccatca gcgcatgcgt ggaacctttg tggctcctct 420

gccgatccat actgcggaac tcctagccgc ttgttttgct cgcagccggt ctggagcaaa 480

gctcatagga actgacaatt ctgtcgtcct ctcgcggaaa tatacatcgt ttc 533

<210>9

<211>158

<212>DNA

<213> Simian cytomegalovirus

<400>9

gtcagacaga cagacagtta tatgggctgg tccctataac tctgccattg taaccccata 60

tagccagaca gttagcattg catctattga tgatgtacta atgtattgta acccccccta 120

tgccattgtc taactgtact aatgtatgat attatacc 158

<210>10

<211>131

<212>DNA

<213> Rabbit (Oryctolagus cuniculus)

<400>10

gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 60

tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 120

tcactcggaa g 131

<210>11

<211>204

<212>DNA

<213> Simian cytomegalovirus

<400>11

atatatactc tatgttatac tctatgatat acaatatata ctcatgaaca ctatgtactt 60

ggtgtatgac tcattattgt ctgggacttg gttgggactt ggttggttgg gaagaatgtt 120

gtgcctgtac ttgtgctgtg ctgtggatct caataaatgt gactatgttc aaaacactaa 180

gtgcccccgt gtcttcttta acta 204

<210>12

<211>163

<212>DNA

<213> herpes simplex virus 2

<400>12

gaagacgagc tctaagggag gggaggggag ctgggcttgt gtataaataa aaagacaccg 60

atgttcaaaa atacacatga cttctggtat tgttttgcct tggtttttat ttgggggggg 120

gggggcgtgt gactagaaaa acaaatgcag acatgtgcta acg 163

<210>13

<211>191

<212>DNA

<213> human papilloma virus type 16

<400>13

aattgttaca tataattgtt gtataccata acttactatt ttttcttttt tattttcata 60

tataattttt ttttttgttt gtttgtttgt tttttaataa actgttatta cttaacaatg 12

cgacacaaac gttctgcaaa acgcacaaaa cgtgcatcgg ctacccaact ttataaaaca 180

tgcaaacagg c 191

<210>14

<211>3759

<212>DNA

<213> Artificial sequence

<220>

<223> pJV expression vector

<220>

<221> Intron

<222>(1725)..(1857)

<223> rat insulin intron A

<220>

<221>misc_feature

<222>(1)..(44)

<223> Tn903, pUC4K residue

<220>

<221>misc_feature

<222>(861)..(896)

<223> Tn903, pUC4K residue

<220>

<221>misc_feature

<222>(897)..(902)

<223>pUC19MCS

<220>

<221> polyA Signal

<222>(2556)..(2686)

<223>rGLOB pA

<220>

<221> polyA site

<222>(2647)..(2647)

<223> PolyA _ site _1

<220>

<221> promoter

<222>(903)..(1587)

<223>CMV Pro

<220>

<221>3′UTR

<222>(2012)..(2544)

<223>HBVenh

<220>

<221>5′UTR

<222>(1864)..(1984)

<223> 5' -UTR of HBV pre-S2

<220>

<221>misc_feature

<222>(1719)..(1724)

<223> Bam/Bgl fusion

<220>

<221>misc_feature

<222>(1985)..(1987)

<223>ATG-Nhe

<220>

<221>misc_feature

<222>(1988)..(2011)

<223> CDS insertion site

<220>

<221>misc_feature

<222>(2545)..(2555)

<223> unknown

<220>

<221> exon

<222>(1588)..(1718)

<223> CMV exon 1/2

<220>

<221>misc_feature

<222>(2693)..(3759)

<223>pUC19

<220>

<221>misc_feature

<222>(45)..(860)

<223> KanR (Tn903) complementary sequence

<400>14

ggcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 60

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 120

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 180

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 240

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 300

ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 360

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 420

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 480

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 540

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 600

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 660

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 720

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 780

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 840

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacaggtcg 900

acaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata 960

ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt 1020

aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 1080

cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 1140

cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 1200

tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 1260

ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 1320

actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 1380

tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 1440

accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 1500

gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 1560

atataagcag agctcgttta gtgaacc gtc aga tcg cct gga gac gcc atc cac 1614

Val Arg Ser Pro Gly Asp Ala Ile His

15

gct gtt ttg acc tcc ata gaa gac acc ggg acc gat cca gcc tcc gcg 1662

Ala Val Leu Thr Ser Ile Glu Asp Thr Gly Thr Asp Pro Ala Ser Ala

10 15 20 25

gcc ggg aac ggt gca ttg gaa cgc gga ttc ccc gtg cca aga gtg act 1710

Ala Gly Asn Gly Ala Leu Glu Arg Gly Phe Pro Val Pro Arg Val Thr

30 35 40

cac cgt cc ggatctcagc aagcaggtat gtactctcca gggtgggcct ggcttcccca 1768

His Arg

gtcaagactc cagggatttg agggacgctg tgggctcttc tcttacatgt accttttgct 1828

tgcctcaacc ctgactatct tccaggtcag gatcccagag tcaggggtct gtattttcct 1888

gctggtggct ccagttcagg aacagtaaac cctgctccga atattgcctc tcacatctcg 1948

tcaatctccg cgaggactgg ggaccctgtg acgaacatgg ctagcgggcc cagatctggg 2008

ccctaacaaa acaaaaagat ggggttattc cctaaacttc atgggttacg taattggaag 2068

ttgggggaca ttgccacaag atcatattgt acaaaagatc aaacactgtt ttagaaaact 2128

tcctgtaaac aggcctattg attggaaagt atgtcaaagg attgtgggtc ttttgggctt 2188

tgctgctcca tttacacaat gtggatatcc tgccttaatg cctttgtatg catgtataca 2248

agctaaacag gctttcactt tctcgccaac ttacaaggcc tttctaagta aacagtacat 2308

gaacctttac cccgttgctc ggcaacggcc tggtctgtgc caagtgtttg ctgacgcaac 2368

ccccactggc tggggcttgg ccataggcca tcagcgcatg cgtggaacct ttgtggctcc 2428

tctgccgatc catactgcgg aactcctagc cgcttgtttt gctcgcagcc ggtctggagc 2488

aaagctcata ggaactgaca attctgtcgt cctctcgcgg aaatatacat cgtttcgatc 2548

tacgtatgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca 2608

tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt 2668

gtgtctctca ctcggaagga attctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 2728

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 2788

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 2848

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 2908

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 2968

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3028

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3088

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3148

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3208

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3268

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3328

agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 3388

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3448

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 3508

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 3568

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 3628

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 3688

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 3748

ttgcctgact c 3759

<210>15

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>15

ggaggatccg gacggtgagt cactcttggc acggggaatc cg 42

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>16

ggtgaatatg gctcataaca c 21

<210>17

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>17

ccgccgaaca tggagaacat cgc 23

<210>18

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>18

cacagatctt ttgttagggt ttaaatgtat acc 33

<210>19

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>19

ggaggatcct gacctggaag atagtcacc 29

<210>20

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>20

ggaggatcca tcagcaagca ggtatg 26

<210>21

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>21

ggagctagcg ggcgtttgac ctccggcgtc ggg 33

<210>22

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>22

ggagaattca gatctcctct agtaaaacaa tggctgg 37

<210>23

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>23

ggagctagcc ttctaaccga ggtcg 25

<210>24

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>24

ggaagatctc cttactccag ctctatgctg 30

<210>25

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>25

ggcgaattcc ttccgagtga gagacac 27

<210>26

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>26

ggagtataca tttaaagggc cctaacaaaa caaaaagatg ggg 43

<210>27

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>27

ggagctagct cgtttacttt gaccaagaac g 31

<210>28

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>28

ggaagatctc cttatttttg acaccagacc aactgg 36

<210>29

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>29

ggagtcgacc tgtctgctta cataaacag 29

<210>30

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>30

cgtaatgctc tgccagtgtt acaacc 26

<210>31

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>31

gaaagatctc agcaagcagg 20

<210>32

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>32

ggaggatcct gacctggaag atagtcaggg ttgaggcaag caaaagg 47

<210>33

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>33

ctagcgggcc ca 12

<210>34

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>34

gatctgggcc cg 12

<210>35

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>35

ggagctagca tcatcccagt tgaggagg 28

<210>36

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>36

ggtagatctc ctcatgtctg ctcgaagc 28

<210>37

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>37

ccaagctagc gacaaaactc acacatgcc 29

<210>38

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>38

ggaagatctc gtttacccct gtcatttacc cggagacagg gagag 45

<210>39

<211>57

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400>39

aagatgtcca gactctgtct ctccgtggcc ctcctcgtgc tcctcgggac actcgcc 57

<210>40

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>40

ggaactagta agatgtccag actc 24

<210>41

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>41

ggaagctagc ggcgagtgtc ccgag 25

<210>42

<211>75

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400>42

ggaaagatgg ccagcctctt tgccacattt ctcgtggtgc tcgtgagcct cagcctcgcc 60

agcgaaagca gcgcc 75

<210>43

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>43

ggaactagtg gaaagatggc cagc 24

<210>44

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>44

ggaagctagc ggcgctgctt tcgctg 26

<210>45

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide

<400>45

aggtctttgc taatcttggt gctttgcttc ctgcccctgg ctgctctggg g 51

<210>46

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>46

ggaactagta ggtctttgct aatc 24

<210>47

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>47

ggaagctagc ccccagagca gccag 25

<210>48

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>48

ggagctagct cgtttacttt gaccaagaac g 31

<210>49

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>49

ggaagatctc cggtgagtgg tgctg 25

<210>50

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>50

gcaggatcca gtagacctgg agagaggaca ag 32

<210>51

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>51

ggaagatcta caaggtgagc tgctgtggc 29

<210>52

<211>490

<212>DNA

<213> Pseudorabies Virus

<400>52

tggccgcaga gcgggccggg catgcaaatc agaggcgcgc gggagacgcc tccgcgcgcc 60

cattggcccg ggcgagccga gatggccgcc gcgggggccg gacatgcaaa gtagacgcga 120

gaggaagtag ggagagaaat cccattggcc gtcgaggggc caagatggcg ccctcggggc 180

cggacatgca aagtagacgc gagaggaagt gggcgagaga aatcccattg gccgtcgatg 240

gggcaagatg gccgccgcgg gggccgggca tgcaaatggt cctcgcgagg aagttcctcg 300

cgaaatccca ttggccggcg gccgccatct tgggccgggc atgcaaagca gacggcagag 360

gaagcgggcg agaaaaatcc cattggccgg ccgtcgggga agtccgcggc gaaaatcggc 420

cattggtccg cttacctggg ggcgggctct cctcggggcg cttataagcg cggtctccat 480

cgtagcactt 490

<210>53

<211>495

<212>DNA

<213> Rous sarcoma virus

<400>53

ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 60

acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 120

ctgcttcgcg atgtacgggc cagatatacg cgtatctgag gggactaggg tgtgtttagg 180

cgaaaagcgg ggcttcggtt gtacgcggtt aggagttccc tcaggatata gtagtttcgc 240

ttttgcatag ggagggggaa atgtagtctt atgcaataca cttgtagtct tgcaacatgg 300

taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg 360

tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacaggt ctgacatgga 420

ttggacgaac cactgaattc cgcattgcag agataattgt atttaagtgc ctagctcgat 480

acaataaacg ccatt 495

<210>54

<211>5446

<212>DNA

<213> Artificial sequence

<220>

<221> kanamycin resistance gene flanked by Tn903 sequences

<222>(1)..(896)

<220>

<221> SalI cloning site

<222>(897)..(902)

<220>

<221> CMV promoter

<222>(903)..(1587)

<220>

<221> CMV exon 1/2 fusion

<222>(1588)..(1718)

<220>

<221> Bam H1/Bgl2 fusion

<222>(1719)..(1724)

<220>

<221> rat insulin intron A

<222>(1725)..(1857)

<220>

<221> Bam H1 cloning site

<222>(1858)..(1863)

<220>

<221> pre S2 region of HbsAg non-coding region

<222>(1864)..(1984)

<220>

<221>CDS

<222>(1985)..(3691)

<220>

G nucleotide of <221> H3Panama 3' UTR

<222>(3692)..(3692)

<220>

<221> Bsp1201 cloning site

<222>(3693)..(3698)

<220>

<221> HBV enhancer

<222>(3699)..(4231)

<220>

<221> rabbit beta globin polyadenylation region

<222>(4243)..(4373)

<220>

<221> Eco RI cloning site

<222>(4374)..(4379)

<220>

<221> pUC 19 vector backbone sequence

<222>(4380)..(5446)

<400>54

ggcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 60

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 120

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 180

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 240

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 300

ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 360

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 420

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcaaatgcaa ccggcgcagg 480

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 540

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 600

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 660

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 720

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 780

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 840

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacaggtcg 900

acaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata 960

ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt 1020

aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 1080

cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 1140

cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 1200

tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 1260

ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 1320

actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 1380

tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 1440

accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 1500

gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 1560

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 1620

ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg 1680

gaacgcggat tccccgtgcc aagagtgact caccgtccgg atctcagcaa gcaggtatgt 1740

actctccagg gtgggcctgg cttccccagt caagactcca gggatttgag ggacgctgtg 1800

ggctcttctc ttacatgtac cttttgcttg cctcaaccct gactatcttc caggtcagga 1860

tcccagagtc aggggtctgt attttcctgc tggtggctcc agttcaggaa cagtaaaccc 1920

tgctccgaat attgcctctc acatctcgtc aatctccgcg aggactgggg accctgtgac 1980

gaac atg gct agc aag act atc att gct ttg agc tac att tta tgt ctg 2029

Met Ala Ser Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu

1 5 10 15

gtt ttc gct caa aaa ctt ccc gga aat gac aac agc acg gca acg ctg 2077

Val Phe Ala Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu

20 25 30

tgc ctg ggg cac cat gca gtg tca aac gga acg cta gtg aaa aca atc 2125

Cys Leu Gly His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile

35 40 45

acg aat gac caa att gaa gtg act aat gct act gag ctg gtt cag agt 2173

Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser

50 55 60

tcc tca aca ggt aga ata tgc gac agt cct cac caa atc ctt gat gga 2221

Ser Ser Thr Gly Arg Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly

65 70 75

gaa aac tgc aca cta ata gat gct cta ttg gga gac cct cat tgt gat 2269

Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp

80 85 90 95

ggc ttc caa aat aag gaa tgg gac ctt ttt gtt gaa cgc agc aaa gcc 2317

Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala

100 105 110

tac agc aac tgt tac cct tat gat gtg ccg gat tat gcc tcc ctt agg 2365

Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg

115 120 125

tca cta gtt gcc tca tcc ggc aca ctg gag ttt aac aat gaa agc ttc 2413

Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe

130 135 140

aat tgg act gga gtc gct cag aat gga aca agc tct gct tgc aaa agg 2461

Asn Trp Thr Gly Val Ala Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg

145 150 155

aga tct aat aaa agt ttc ttt agt aga ttg aat tgg ttg cac caa tta 2509

Arg Ser Asn Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu His Gln Leu

160 165 170 175

aaa tac aaa tat cca gca ctg aac gtg act atg cca aac aat gaa aaa 2557

Lys Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys

180 185 190

ttt gac aaa ttg tac att tgg ggg gtt ctc cac ccg agt acg gac agt 2605

Phe Asp Lys Leu Tyr Ile Trp Gly Val Leu His Pro Ser Thr Asp Ser

195 200 205

gac caa atc agc cta tat gct caa gca tca ggg aga gtc aca gtc tct 2653

Asp Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Val Thr Val Ser

210 215 220

acc aaa aga agc caa caa act gta atc ccg aat atc gga tct aga ccc 2701

Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser ArgPro

225 230 235

tgg gta agg ggt gtc tcc agc aga ata agc atc tat tgg aca ata gta 2749

Trp Val Arg Gly Val Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val

240 245 250 255

aaa ccg gga gac ata ctt ttg att aac agc aca ggg aat cta att gct 2797

Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala

260 265 270

cct cgg ggt tac ttc aaa ata cga agt ggg aaa agc tca ata atg agg 2845

Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg

275 280 285

tca gat gca ccc att ggc aaa tgc aat tct gaa tgc atc act cca aat 2893

Ser Asp Ala pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn

290 295 300

gga agc att ccc aat gac aaa cca ttt caa aat gta aac agg atc aca 2941

Gly Ser Ile pro Asn Asp Lys Pro Phe Gln Asn Val Asn Ar gIle Thr

305 310 315

tat ggg gcc tgt ccc aga tat gtt aag caa aac act ctg aaa ttg gca 2989

Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala

320 325 330 335

aca ggg atg cgg aat gta cca gag aaa caa act aga ggc ata ttc ggc 3037

Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly

340 345 350

gca atc gcg ggt ttc ata gaa aat ggt tgg gag gga atg gtg gac ggt 3085

Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly

355 360 365

tgg tac ggt ttc agg cat caa aat tct gag ggc aca gga caa gca gca 3133

Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala

370 375 380

gat ctt aaa agc act caa gca gca atc aac caa atc aac ggg aaa ctg 3181

Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu

385 390 395

aat agg tta atc gag aaa acg aac gag aaa ttc cat caa att gaa aaa 3229

Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys

400 405 410 415

gaa ttc tca gaa gta gaa ggg aga att cag gac ctc gag aaa tat gtt 3277

Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val

420 425 430

gag gac act aaa ata gat ctc tgg tcg tac aac gcg gag ctt ctt gtt 3325

Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val

435 440 445

gcc ctg gag aac caa cat aca att gat cta act gac tca gaa atg aac 3373

Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn

450 455 460

aaa ctg ttt gaa aga aca aag aag caa ctg agg gaa aat gct gag gat 3421

Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp

465 470 475

atg ggc aat ggt tgt ttc aaa ata tac cac aaa tgt gac aat gcc tgc 3469

Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys

480 485 490 495

ata ggg tca atc aga aat gga act tat gac cat gat gta tac aga gac 3517

Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp

500 505 510

gaa gca tta aac aac cgg ttc cag atc aaa ggt gtt gag ctg aag tca 3565

Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser

515 520 525

gga tac aaa gat tgg atc cta tgg att tcc ttt gcc ata tca tgc ttt 3613

Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe

530 535 540

ttg ctt tgt gtt gtt ttg ctg ggg ttc atc atg tgg gcc tgc caa aaa 3661

Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys

545 550 555

ggc aac att agg tgc aac att tgc att tga ggggccctaa caaaacaaaa 3711

Gly Asn Ile Arg Cys Asn Ile Cys Ile

560 565

agatggggtt attccctaaa cttcatgggt tacgtaattg gaagttgggg gacattgcca 3771

caagatcata ttgtacaaaa gatcaaacac tgttttagaa aacttcctgt aaacaggcct 3831

attgattgga aagtatgtca aaggattgtg ggtcttttgg gctttgctgc tccatttaca 3891

caatgtggat atcctgcctt aatgcctttg tatgcatgta tacaagctaa acaggctttc 3951

actttctcgc caacttacaa ggcctttcta agtaaacagt acatgaacct ttaccccgtt 4011

gctcggcaac ggcctggtct gtgccaagtg tttgctgacg caacccccac tggctggggc 4071

ttggccatag gccatcagcg catgcgtgga acctttgtgg ctcctctgcc gatccatact 4131

gcggaactcc tagccgcttg ttttgctcgc agccggtctg gagcaaagct cataggaact 4191

gacaattctg tcgtcctctc gcggaaatat acatcgtttc gatctacgta tgatcttttt 4251

ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa 4311

taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga 4371

aggaattctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 4431

ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 4491

atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 4551

gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 4611

gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 4671

gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 4731

gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 4791

aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4851

ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4911

taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4971

tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 5031

gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 5091

taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 5151

tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5211

tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 5271

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgcaagttt 5331

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 5391

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactc 5446

<210>55

<211>568

<212>PRT

<213> Artificial sequence

<400>55

Met Ala Ser Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val

1 5 10 15

Phe Ala Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys

20 25 30

Leu Gly His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr

35 40 45

Asn Asp GlnIle Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser

50 55 60

Ser Thr Gly Arg Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu

65 70 75 80

Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly

85 90 95

Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr

100 105 110

Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser

115 120 125

Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn

130 135 140

Trp Thr Gly Val Ala Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg

145 150 155 160

Ser Asn Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu His Gln Leu Lys

165 170 175

Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe

180 185 190

Asp Lys Leu Tyr Ile Trp Gly Val Leu His Pro Ser Thr Asp Ser Asp

195 200 205

Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Val Thr Val Ser Thr

210 215 220

Lys Arg Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Trp

225 230 235 240

Val Arg Gly Val Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys

245 250 255

Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro

260 265 270

Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser

275 280 285

Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly

290 295 300

Ser Ile pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr

305 310 315 320

Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr

325 330 335

Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala

340 345 350

Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp

355 360 365

Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp

370 375 380

Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn

385 390 395 400

Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu

405 410 415

Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu

420 425 430

Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala

435 440 445

Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys

450 455 460

Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met

465 470 475 480

Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile

485 490 495

Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu

500 505 510

Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly

515 520 525

Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu

530 535 540

Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly

545 550 555 560

Asn Ile Arg Cys Asn Ile Cys Ile

565

<210>56

<211>63

<212>PRT

<213> Artificial sequence

<220>

<223> N-terminal peptide of H3Panama HA influenza virus antigen

<400>56

Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala

1 5 10 15

Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly

20 25 30

His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp

35 40 45

Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser

50 55 60

<210>57

<211>65

<212>PRT

<213> Artificial sequence

<220>

<223> N-terminal sequence of H3Panama HA antigen encoded by pPJV1671

<400>57

Met Ala Ser Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val

1 5 10 15

Phe Ala Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys

20 25 30

Leu Gly His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr

35 40 45

Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser

50 55 60

Ser

65

<210>58

<211>62

<212>PRT

<213> Artificial sequence

<220>

<223> consensus sequence of native and ppJV 1671N-terminal H3 Panama HA antigens

<400>58

Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gln

1 5 10 15

Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His

20 25 30

His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gln

35 40 45

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser

50 55 60

<210>59

<211>5453

<212>DNA

<213> Artificial sequence

<220>

<221> kanamycin resistance gene flanked by Tn903 sequences

<222>(1)..(896)

<220>

<221> SalI cloning site

<222>(897)..(902)

<220>

<221> CMV start

<222>(903)..(1587)

<220>

<221> CMV exon 1/2 fusion

<222>(1588)..(1718)

<220>

<221> Bam H1/Bg12 fusion

<222>(1719)..(1724)

<220>

<221> rat insulin intron A

<222>(1725)..(1857)

<220>

<221> Bam H1 cloning site

<222>(1858)..(1863)

<220>

<221> pre S2 region of HBsAg non-coding region

<222>(1864)..(1984)

<220>

<221>CDS

<222>(1985)..(3697)

<220>

<221> Bsp1201 cloning site

<222>(3700)..(3705)

<220>

<221> HBV enhancer

<222>(3706)..(4238)

<220>

<221> rabbit beta globin polyadenylation region

<222>(4250)..(4380)

<220>

<221> Eco RI cloning site

<222>(4381)..(4386)

<220>

<221> pUC 19 vector backbone sequence

<222>(4387)..(5453)

<400>59

ggcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 60

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 120

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 180

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 240

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 300

ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 360

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 420

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 480

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 540

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 600

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 660

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 720

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 780

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 840

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacaggtcg 900

acaatattgg ctattggcca ttgcatacgt tgtatctata tcataatatg tacatttata 960

ttggctcatg tccaatatga ccgccatgtt gacattgatt attgactagt tattaatagt 1020

aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 1080

cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 1140

cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 1200

tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta 1260

ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg 1320

actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 1380

tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 1440

accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 1500

gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 1560

atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 1620

ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg 1680

gaacgcggat tccccgtgcc aagagtgact caccgtccgg atctcagcaa gcaggtatgt 1740

actctccagg gtgggcctgg cttccccagt caagactcca gggatttgag ggacgctgtg 1800

ggctcttctc ttacatgtac cttttgcttg cctcaaccct gactatcttc caggtcagga 1860

tcccagagtc aggggtctgt attttcctgc tggtggctcc agttcaggaa cagtaaaccc 1920

tgctccgaat attgcctctc acatctcgtc aatctccgcg aggactgggg accctgtgac 1980

gaac atg gct agc gag aaa ata gtg ctt ctt ttt gca ata gtc agt ctt 2029

Met Ala Ser Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu

1 5 10 15

gtt aaa agt gat cag att tgc att ggt tac cat gca aac aac tcg aca 2077

Val Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr

20 25 30

gag cag gtt gac aca ata atg gaa aag aac gtt act gtt aca cat gcc 2125

Glu Gln yal Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala

35 40 45

caa gac ata ctg gaa aag aca cac aat ggg aag ctc tgc gat cta gat 2173

Gln Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp

50 55 60

gga gtg aag cct cta att ttg aga gat tgt agt gta gct gga tgg ctc 2221

Gly Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu

65 70 75

ctc gga aac cca atg tgt gac gaa ttc atc aat gtg ccg gaa tgg tct 2269

Leu Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser

80 85 90 95

tac ata gtg gag aag gcc aat cca gtc aat gac ctc tgt tac cca ggg 2317

Tyr Ile Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly

100 105 110

gat ttc aat gac tat gaa gaa ttg aaa cac cta ttg agc aga ata aac 2365

Asp Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn

115 120 125

cat ttt gag aaa att cag atc atc ccc aaa agt tct tgg tcc agt cat 2413

His Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His

130 135 140

gaa gcc tca ttg ggg gtg agc tca gca tgt cca tac cag gga aag tcc 2461

Glu Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser

145 150 155

tcc ttt ttc aga aat gtg gta tgg ctt atc aaa aag aac agt aca tac 2509

Ser Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr

160 165 170 175

cca aca ata aag agg agc tac aat aat acc aac caa gaa gat ctt ttg 2557

Pro Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu

180 185 190

gta ctg tgg ggg att cac cat cct aat gat gcg gca gag cag aca aag 2605

Val Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys

195 200 205

ctc tat caa aac cca acc acc tat att tcc gtt ggg aca tca aca cta 2653

Leu Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu

210 215 220

aac cag aga ttg gta cca aga ata gct act aga tcc aaa gta aac ggg 2701

Asn Gln Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly

225 230 235

caa agt gga agg atg gag ttc ttc tgg aca att tta aaa ccg aat gat 2749

Gln Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp

240 245 250 255

gca atc aac ttc gag agt aat gga aat ttc att gct cca gaa tat gca 2797

Ala Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala

260 265 270

tac aaa att gtc aag aaa ggg gac tca aca att atg aaa agt gaa ttg 2845

Tyr Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu

275 280 285

gaa tat ggt aac tgc aac acc aag tgt caa act cca atg ggg gcg ata 2893

Glu Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile

290 295 300

aac tct agc atg cca ttc cac aat ata cac cct ctc acc atc ggg gaa 2941

Asn Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu

305 310 315

tgc ccc aaa tat gtg aaa tca aac aga tta gtc ctt gcg act ggg ctc 2989

Cys Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu

320 325 330 335

aga aat agc cct caa aga gag aga aga aga aaa aag aga gga tta ttt 3037

Arg Asn Ser Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe

340 345 350

gga gct ata gca ggt ttt ata gag gga gga tgg cag gga atg gta gat 3085

Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp

355 360 365

ggt tgg tat ggg tac cac cat agc aac gag cag ggg agt ggg tac gct 3133

Gly Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala

370 375 380

gca gac aaa gaa tcc act caa aag gca ata gat gga gtc acc aat aag 3181

Ala Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys

385 390 395

gtc aac tcg att att gac aaa atg aac act cag ttt gag gcc gtt gga 3229

Val Asn Ser Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly

400 405 410 415

agg gaa ttt aac aac tta gaa agg aga ata gag aat tta aac aag aag 3277

Arg Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys

420 425 430

atg gaa gac ggg ttc cta gat gtc tgg act tat aat gct gaa ctt cta 3325

Met Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu

435 440 445

gtt ctc atg gaa aac gag aga act cta gac ttt cat gac tca aat gtc 3373

Val Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val

450 455 460

aag aac ctt tac gac aag gtc cga cta cag ctt agg gat aat gca aag 3421

Lys Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys

465 470 475

gag ctg ggt aac ggt tgt ttc gag ttc tat cat aaa tgt gat aat gaa 3469

Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu

480 485 490 495

tgt atg gaa agt gta aga aac gga acg tat gac tac ccg cag tat tca 3517

Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser

500 505 510

gaa gaa gca aga cta aaa aga gag gaa ata agt gga gta aaa ttg gaa 3565

Glu Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu

515 520 525

tca ata gga att tac caa ata ttg tca att tat tct aca gtg gcg agc 3613

Ser Ile Gly Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser

530 535 540

tcc cta gca ctg gca atc atg gta gct ggt cta tcc tta tgg atg tgc 3661

Ser Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys

545 550 555

tcc aat ggg tcg tta caa tgc aga att tgc att taa atgggcccta 3707

Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

560 565 570

acaaaacaaa aagatggggt tattccctaa acttcatggg ttacgtaatt ggaagttggg 3767

ggacattgcc acaagatcat attgtacaaa agatcaaaca ctgttttaga aaacttcctg 3827

taaacaggcc tattgattgg aaagtatgtc aaaggattgt gggtcttttg ggctttgctg 3887

ctccatttac acaatgtgga tatcctgcct taatgccttt gtatgcatgt atacaagcta 3947

aacaggcttt cactttctcg ccaacttaca aggcctttct aagtaaacag tacatgaacc 4007

tttaccccgt tgctcggcaa cggcctggtc tgtgccaagt gtttgctgac gcaaccccca 4067

ctggctgggg cttggccata ggccatcagc gcatgcgtgg aacctttgtg gctcctctgc 4127

cgatccatac tgcggaactc ctagccgctt gttttgctcg cagccggtct ggagcaaagc 4187

tcataggaac tgacaattct gtcgtcctct cgcggaaata tacatcgttt cgatctacgt 4247

atgatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga 4307

cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc 4367

tctcactcgg aaggaattct gcattaatga atcggccaac gcgcggggag aggcggtttg 4427

cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4487

cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4547

aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4607

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4667

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4727

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4787

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4847

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4907

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4967

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 5027

ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 5087

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 5147

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 5207

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 5267

taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 5327

aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 5387

tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 5447

tgactc 5453

<210>60

<211>570

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400>60

Met Ala Ser Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val

1 5 10 15

Lys Ser Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu

20 25 30

Gln Val Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln

35 40 45

Asp Ile Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly

50 55 60

Val Lys Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu

65 70 75 80

Gly Asn Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr

85 90 95

Ile Val Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp

100 105 110

Phe Asn Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His

115 120 125

Phe Glu Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu

130 135 140

Ala Ser Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser

145 150 155 160

Phe Phe Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro

165 170 175

Thr Ile Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val

180 185 190

Leu Trp Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu

195 200 205

Tyr Gln Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn

2l0 215 220

Gln Arg Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln

225 230 235 240

Ser Gly Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala

245 250 255

Ile Asn Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr

260 265 270

Lys Ile Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu

275 280 285

Tyr Gly Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn

290 295 300

Ser Ser Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys

305 310 315 320

Pro Lys Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg

325 330 335

Asn Ser Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe Gly

340 345 350

Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly

355 360 365

Trp Tyr Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala

370 375 380

Asp Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val

385 390 395 400

Asn Sen Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg

405 410 415

Glu Phe Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met

420 425 430

Glu Asp Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val

435 440 445

Leu Met Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys

450 455 460

Asn Leu Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu

465 470 475 480

Leu Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys

485 490 495

Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu

500 505 510

Glu Ala Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser

515 520 525

Ile GlyIle Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser

530 535 540

Leu Ala Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser

545 550 555 560

Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

565 570

<210>61

<211>5578

<212>DNA

<213> Artificial construct

<220>

<223> PJV2012 construct containing LT A and LT B coding sequences

<400>61

ggcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 60

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 120

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 180

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 240

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 300

ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 360

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 420

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcaaatgcaa ccggcgcagg 480

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 540

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 600

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 660

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 720

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 780

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 840

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacaggtcg 900

acaattcctt ccgagtgaga gacacaaaaa attccaacac actattgcaa tgaaaataaa 960

tttcctttat tagccagaag tcagatgctc aaggggcttc atgatgtccc cataattttt 1020

ggcagaggga aaaagatccc tagttttcca tactgattgc cgcaattgaa ttgggggttt 1080

tattattcca tacacataat ttatcaattt tggtctcggt cagatatgtg attcttaatg 1140

tgtccttcat cctttcaatg gctttttttt gggagtctat atgttgactg cccgggactt 1200

cgacctgaaa tgttgcgccg ctcttaaatg taatgataac catttctctt ttgcctgcca 1260

tcgattccgt atatgatagt atcttgtcat ttatcgtata tatttgtgtg ttgcgatatt 1320

ccgaacatag ttctgtaata gactggggag cgctagcccc cagagcagcc aggggcagga 1380

agcaaagcac caagattagc aaagacctac tagccatgtt cgtcacaggg tccccagtcc 1440

tcgcggagat tgacgagatg tgagaggcaa tattcggagc agggtttact gttcctgaac 1500

tggagccacc agcaggaaaa tacagacccc tgactctggg atcctgacct ggaagatagt 1560

cagggttgag gcaagcaaaa ggtacatgta agagaagagc ccacagcgtc cctcaaatcc 1620

ctggagtctt gactggggaa gccaggccca ccctggagag tacatacctg cttgctgaga 1680

tccggacggt gagtcactct tggcacgggg aatccgcgtt ccaatgcacc gttcccggcc 1740

gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct 1800

ccaggcgatc tgacggttca ctaaacgagc tctgcttata tagacctccc accgtacacg 1860

cctaccgccc atttgcgtca acggggcggg gttattacga cattttggaa agtcccgttg 1920

attttggtgc tcgacctgca ggtcgacaat attggctatt ggccattgca tacgttgtat 1980

ctatatcata atatgtacat ttatattggc tcatgtccaa tatgaccgcc atgttgacat 2040

tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 2100

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 2160

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 2220

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 2280

tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 2340

tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta cgtattagtc 2400

atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg atagcggttt 2460

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 2520

caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc 2580

ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 2640

gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc 2700

ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag tgactcaccg 2760

tccggatctc agcaagcagg tatgtactct ccagggtggg cctggcttcc ccagtcaaga 2820

ctccagggat ttgagggacg ctgtgggctc ttctcttaca tgtacctttt gcttgcctca 2880

accctgacta tcttccaggt caggatccca gagtcagggg tctgtatttt cctgctggtg 2940

gctccagttc aggaacagta aaccctgctc cgaatattgc ctctcacatc tcgtcaatct 3000

ccgcgaggac tggggaccct gtgacgaaca tggctagtag gtctttgcta atcttggtgc 3060

tttgcttcct gcccctggct gctctggggg ctagcaatgg cgacaaatta taccgtgctg 3120

actctagacc cccagatgaa ataaaacgtt ccggaggtct tatgcccaga gggcataatg 3180

agtacttcga tagaggaact caaatgaata ttaatcttta tgatcacgcg agaggaacac 3240

aaaccggctt tgtcagatat gatgacggat atgtttccac ttctcttagt ttgagaagtg 3300

ctcacttagc aggacagtct atattatcag gatattccac ttactatata tatgttatag 3360

cgacagcacc aaatatgttt aatgttaatg atgtattagg cgtatacagc cctcacccat 3420

atgaacagga ggtttctgcg ttaggtggaa taccatattc tcagatatat ggatggtatc 3480

gtgttaattt tggtgtgatt gatgaacgat tacatcgtaa cagggaatat agagaccggt 3540

attacagaaa tctgaatata gctccggcag aggatggtta cagattagca ggtttcccac 3600

cggatcacca agcttggaga gaagaaccct ggattcatca tgcaccacaa ggttgtggaa 3660

attcatcaag aacaattaca ggtgatactt gtaatgagga gacccagaat ctgagcacaa 3720

tatatctcag gaaatatcaa tcaaaagtta agaggcagat attttcagac tatcagtcag 3780

aggttgacat atataacaga attcgggatg aattatgagg atctgggccc taacaaaaca 3840

aaaagatggg gttattccct aaacttcatg ggttacgtaa ttggaagttg ggggacattg 3900

ccacaagatc atattgtaca aaagatcaaa cactgtttta gaaaacttcc tgtaaacagg 3960

cctattgatt ggaaagtatg tcaaaggatt gtgggtcttt tgggctttgc tgctccattt 4020

acacaatgtg gatatcctgc cttaatgcct ttgtatgcat gtatacaagc taaacaggct 4080

ttcactttct cgccaactta caaggccttt ctaagtaaac agtacatgaa cctttacccc 4140

gttgctcggc aacggcctgg tctgtgccaa gtgtttgctg acgcaacccc cactggctgg 4200

ggcttggcca taggccatca gcgcatgcgt ggaacctttg tggctcctct gccgatccat 4260

actgcggaac tcctagccgc ttgttttgct cgcagccggt ctggagcaaa gctcatagga 4320

actgacaatt ctgtcgtcct ctcgcggaaa tatacatcgt ttcgatctac gtatgatctt 4380

tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc 4440

taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc 4500

ggaaggaatt ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 4560

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 4620

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 4680

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4740

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4800

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 4860

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 4920

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 4980

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5040

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5100

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5160

gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5220

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 5280

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 5340

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 5400

tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5460

ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5520

cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactc 5578

<210>62

<211>6254

<212>DNA

<213> Artificial sequence

<220>

<223> PJV7788 construct

<400>62

ggcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 60

agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 120

agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 180

tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 240

tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 300

ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 360

tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 420

aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 480

aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 540

aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 600

aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 660

tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 720

ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 780

ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 840

tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 900

attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 960

acgtggcttt cccccccccc ccggcatgcc tgcaggtcga caatattggc tattggccat 1020

tgcatacgtt gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac 1080

cgccatgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 1140

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 1200

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 1260

caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 1320

cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat 1380

ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca 1440

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac accaatgggc 1500

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 1560

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt 1620

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag 1680

tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc 1740

gggaccgatc cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca 1800

agagtgactc accgtccgga tctcagcaag caggtatgta ctctccaggg tgggcctggc 1860

ttccccagtc aagactccag ggatttgagg gacgctgtgg gctcttctct tacatgtacc 1920

ttttgcttgc ctcaaccctg actatcttcc aggtcaggat cccagagtca ggggtctgta 1980

ttttcctgct ggtggctcca gttcaggaac agtaaaccct gctccgaata ttgcctctca 2040

catctcgtca atctccgcga ggactgggga ccctgtgacg aacatggcta gtaggtcttt 2100

gctaatcttg gtgctttgct tcctgcccct ggctgctctg ggggctagcg ctccccagtc 2160

tattacagaa ctatgttcgg aatatcgcaa cacacaaata tatacgataa atgacaagat 2220

actatcatat acggaatcga tggcaggcaa aagagaaatg gttatcatta catttaagag 2280

cggcgcaaca tttcaggtcg aagtcccggg cagtcaacat atagactccc aaaaaaaagc 2340

cattgaaagg atgaaggaca cattaagaat cacatatctg accgagacca aaattgataa 2400

attatgtgta tggaataata aaacccccaa ttcaattgcg gcaatcagta tggaaaacta 2460

gggatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga 2520

cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc 2580

tctcactcgg aaggaattga gggccggccc tctgcaggtc gacaatattg gctattggcc 2640

attgcatacg ttgtatctat atcataatat gtacatttat attggctcat gtccaatatg 2700

accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 2760

agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 2820

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 2880

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 2940

ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa 3000

atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta 3060

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acaccaatgg 3120

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 3180

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc 3240

gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 3300

agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 3360

ccgggaccga tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc 3420

caagagtgac tcaccgtccg gatctcagca agcaggtatg tactctccag ggtgggcctg 3480

gcttccccag tcaagactcc agggatttga gggacgctgt gggctcttct cttacatgta 3540

ccttttgctt gcctcaaccc tgactatctt ccaggtcagg atcccagagt caggggtctg 3600

tattttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 3660

cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatggc tagtaggtct 3720

ttgctaatct tggtgctttg cttcctgccc ctggctgctc tgggggctag caatggcgac 3780

aaattatacc gtgctgactc tagaccccca gatgaaataa aacgttccgg aggtcttatg 3840

cccagagggc ataatgagta cttcgataga ggaactcaaa tgaatattaa tctttatgat 3900

cacgcgagag gaacacaaac cggctttgtc agatatgatg acggatatgt ttccacttct 3960

cttagtttga gaagtgctca cttagcagga cagtctatat tatcaggata ttccacttac 4020

tatatatatg ttatagcgac agcaccaaat atgtttaatg ttaatgatgt attaggcgta 4080

tacagccctc acccatatga acaggaggtt tctgcgttag gtggaatacc atattctcag 4140

atatatggat ggtatcgtgt taattttggt gtgattgatg aacgattaca tcgtaacagg 4200

gaatatagag accggtatta cagaaatctg aatatagctc cggcagagga tggttacaga 4260

ttagcaggtt tcccaccgga tcaccaagct tggagagaag aaccctggat tcatcatgca 4320

ccacaaggtt gtggaaattc atcaagaaca attacaggtg atacttgtaa tgaggagacc 4380

cagaatctga gcacaatata tctcaggaaa tatcaatcaa aagttaagag gcagatattt 4440

tcagactatc agtcagaggt tgacatatat aacagaattc gggatgaatt atgaggatct 4500

gggccctaac aaaacaaaaa gatggggtta ttccctaaac ttcatgggtt acgtaattgg 4560

aagttggggg acattgccac aagatcatat tgtacaaaag atcaaacact gttttagaaa 4620

acttcctgta aacaggccta ttgattggaa agtatgtcaa aggattgtgg gtcttttggg 4680

ctttgctgct ccatttacac aatgtggata tcctgcctta atgcctttgt atgcatgtat 4740

acaagctaaa caggctttca ctttctcgcc aacttacaag gcctttctaa gtaaacagta 4800

catgaacctt taccccgttg ctcggcaacg gcctggtctg tgccaagtgt ttgctgacgc 4860

aacccccact ggctggggct tggccatagg ccatcagcgc atgcgtggaa cctttgtggc 4920

tcctctgccg atccatactg cggaactcct agccgcttgt tttgctcgca gccggtctgg 4980

agcaaagctc ataggaactg acaattctgt cgtcctctcg cggaaatata catcgtttcg 5040

atctacgtat gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga 5100

gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt 5160

tttgtgtctc tcactcggaa ggaattctgc attaatgaat cggccaacgc gcggggagag 5220

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5280

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5340

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5400

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 5460

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 5520

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5580

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 5640

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5700

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5760

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5820

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 5880

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5940

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6000

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6060

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6120

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6180

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6240

tagttgcctg actc 6254

<210>63

<211>18

<212>DNA

<213> Artificial sequence

<400>63

gccactctct tccgacac 18

<210>64

<211>19

<212>DNA

<213> Artificial sequence

<400>64

caagaacatc acacggaac 19

<210>65

<211>512

<212>PRT

<213> herpes simplex virus 2

<400>65

Met Ala Thr Asp Ile Asp Met Leu Ile Asp Leu Gly Leu Asp Leu Ser

1 5 10 15

Asp Ser Glu Leu Glu Glu Asp Ala Leu Glu Arg Asp Glu Glu Gly Arg

20 25 30

Arg Asp Asp Pro Glu Ser Asp Ser Ser Gly Glu Cys Ser Ser Ser Asp

35 40 45

Glu Asp Met Glu Asp Pro Cys Gly Asp Gly Gly Ala Glu Ala Ile Asp

50 55 60

Ala Ala Ile Pro Lys Gly Pro Pro Ala Arg Pro Glu Asp Ala Gly Thr

65 70 75 80

Pro Glu Ala Ser Thr Pro Arg Pro Ala Ala Arg Arg Gly Ala Asp Asp

85 90 95

Pro Pro Pro Ala Thr Thr Gly Val Trp Ser Arg Leu Gly Thr Arg Arg

100 105 110

Ser Ala Ser Pro Arg Glu Pro His Gly Gly Lys Val Ala Arg Ile Gln

115 120 125

Pro Pro Ser Thr Lys Ala Pro His Pro Arg Gly Gly Arg Arg Gly Arg

130 135 140

Arg Arg Gly Arg Gly Arg Tyr Gly Pro Gly Gly Ala Asp Ser Thr Pro

145 150 155 160

Asn Pro Arg Arg Arg Val Ser Arg Asn Ala His Asn Gln Gly Gly Arg

165 170 175

His Pro Ala Ser Ala Arg Thr Asp Gly Pro Gly Ala Thr His Gly Glu

180 185 190

Ala Arg Arg Gly Gly Glu Gln Leu Asp Val Ser Gly Gly Pro Arg Pro

195 200 205

Arg Gly Thr Arg Gln Ala Pro Pro Pro Leu Met Ala Leu Ser Leu Thr

210 215 220

Pro Pro His Ala Asp Gly Arg Ala Pro Val Pro Glu Arg Lys Ala Pro

225 230 235 240

Ser Ala Asp Thr Ile Asp Pro Ala Val Arg Ala Val Leu Arg Ser Ile

245 250 255

Ser Glu Arg Ala Ala Val Glu Arg Ile Ser Glu Ser Phe Gly Arg Ser

260 265 270

Ala Leu Val Met Gln Asp Pro Phe Gly Gly Met Pro Phe Pro Ala Ala

275 280 285

Asn Ser Pro Trp Ala Pro Val Leu Ala Thr Gln Ala Gly Gly Phe Asp

290 295 300

Ala Glu Thr Arg Arg Val Ser Trp Glu Thr Leu Val Ala His Gly Pro

305 310 315 320

Ser Leu Tyr Arg Thr Phe Ala Ala Asn Pro Arg Ala Ala Ser Thr Ala

325 330 335

Lys Ala Met Arg Asp Cys Val Leu Arg Gln Glu Asn Leu Ile Glu Ala

340 345 350

Leu Ala Ser Ala Asp Glu Thr Leu Ala Trp Cys Lys Met Cys Ile His

355 360 365

His Asn Leu Pro Leu Arg Pro Gln Asp Pro Ile Ile Gly Thr Ala Ala

370 375 380

Ala Val Leu Glu Asn Leu Ala Thr Arg Leu Arg Pro Phe Leu Gln Cys

385 390 395 400

Tyr Leu Lys Ala Arg Gly Leu Cys Gly Leu Asp Asp Leu Cys Ser Arg

405 410 415

Arg Arg Leu Ser Asp Ile Lys Asp Ile Ala Ser Phe Val Leu Val Ile

420 425 430

Leu Ala Arg Leu Ala Asn Arg Val Glu Arg Gly Val Ser Glu Ile Asp

435 440 445

Tyr Thr Thr Val Gly Val Gly Ala Gly Glu Thr Met His Phe Tyr Ile

450 455 460

Pro Gly Ala Cys Met Ala Gly Leu Ile Glu Ile Leu Asp Thr His Arg

465 470 475 480

Gln Glu Cys Ser Ser Arg Val Cys Glu Leu Thr Ala Ser His Thr Ile

485 490 495

Ala Pro Leu Tyr Val His Gly Lys Tyr Phe Tyr Cys Asn Ser Leu Phe

500 505 510

<210>66

<211>18

<212>PRT

<213> herpes simplex virus 2

<400>66

Arg Val Ser Trp Glu Thr Leu Val Ala His Gly Pro Ser Leu Tyr Arg

1 5 10 15

Thr Phe

<210>67

<211>18

<212>PRT

<213> herpes simplex virus 2

<400>67

Val Ala His Gly Pro Ser Leu Tyr Arg Thr Phe Ala Ala Asn Pro Arg

1 5 10 15

Ala Ala

<210>68

<211>9

<212>PRT

<213> Artificial sequence

<400>68

His Gly Pro Ser Leu Tyr Arg Thr Phe

1 5

<210>69

<211>11

<212>PRT

<213> herpes simplex virus 2

<400>69

Leu Tyr Arg Thr Phe Ala Ala Asn Pro Arg Ala

1 5 10

<210>70

<211>11

<212>PRT

<213> herpes simplex virus 1

<400>70

Leu Tyr Arg Thr Phe Ala Gly Asn Pro Arg Ala

1 5 10

Claims

1. A nucleic acid construct suitable for delivery to a subject to induce an immune response against an influenza virus Hemagglutinin (HA) antigen, the construct comprising:

(i) a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

(ii) a coding sequence operably linked to said chimeric promoter, wherein said coding sequence encodes an influenza virus Hemagglutinin (HA) antigen, an immunogenic fragment thereof, or an immunogenic variant of said antigen or fragment, said variant having at least 80% amino acid homology to said antigen or fragment;

2. The nucleic acid construct of claim 1, wherein said coding sequence encodes more than one of said HA, fragment or immunogenic variant.

3. The nucleic acid construct of claim 2, wherein the coding sequence encodes the HA, fragment or variant of each of three to five different influenza virus strains.

4. The nucleic acid construct of claim 3, wherein the coding sequence encodes the HA, fragment or variant of each of three or four non-influenza virus strains.

5. The nucleic acid construct of any preceding claim, wherein the coding sequence encodes the HA, fragment or variant of an influenza virus strain.

6. Vector particles coated with a nucleic acid construct according to any of the preceding claims.

7. A vector particle coated with at least two different nucleic acid constructs of claim 1, wherein each of said constructs encodes said HA, fragment or variant of a different influenza virus strain.

8. The vector particle of claim 7, which is coated with three to five different said constructs.

9. The vector particle of claim 8, wherein three or four of said constructs encode said HA, fragment or variant of a different non-influenza virus strain.

10. The vector particle of any one of claims 7 to 9, which is coated with said construct encoding said HA, fragment or variant of a pandemic influenza virus strain.

11. The vector particle of any one of claims 6 to 10, further coated with a nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to the promoter, wherein the coding sequence encodes an ADP-ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof both having adjuvant activity and having at least 80% amino acid homology to the subunit or fragment.

12. The vector particle of claim 11, wherein the promoter sequence is a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

13. The vector particle according to claim 11 or 12, wherein the ADP ribosylating bacterial toxin subunit is selected from the group consisting of cholera toxin subunit a, cholera toxin subunit B, escherichia coli heat-labile toxin subunit a, and escherichia coli heat-labile toxin subunit B.

14. The vector particle of any one of claims 11 to 13, wherein said nucleic acid construct comprises two coding sequences, each comprising a different said subunit, fragment or variant.

15. The vector particle of claim 14, wherein the two coding sequences encode cholera toxin subunit a and cholera toxin subunit B, respectively, or escherichia coli heat-labile toxin subunit a and escherichia coli heat-labile toxin subunit B, respectively.

16. The vector particle of any one of claims 11 to 15, wherein the nucleic acid construct further comprises:

(a) a non-translated leader sequence derived from an HBVpreS2 antigen sequence, an HBV e-antigen sequence, or an HSV-type 2gD antigen sequence, and operably linked to the chimeric promoter; and

(b) an enhancer sequence derived from the 3 'untranslated region (UTR) of the HBsAg sequence or the 3' UTR of the simian CMV immediate early gene sequence, operably linked to the chimeric promoter, and located downstream of the coding sequence.

17. The support particle of any one of claims 6 to 16, which is a gold particle.

18. A dose container for a particle-mediated delivery device comprising the coated particles of any one of claims 6 to 17.

19. A particle-mediated delivery device loaded with the coated particle of any one of claims 6 to 17.

20. The particle-mediated delivery device of claim 19 which is a needleless syringe.

21. A nucleic acid construct suitable for delivery to a subject to induce an immune response against an influenza virus Hemagglutinin (HA) antigen, the construct comprising:

(i) a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

22. The nucleic acid construct of claim 21, further comprising:

(iii) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence and operably linked to the chimeric promoter; or

23. The nucleic acid construct of claim 21 or 22, wherein said coding sequence encodes more than one of said HA, fragment or immunogenic variant.

24. The nucleic acid construct of claim 23, wherein the coding sequence encodes the HA, fragment or variant of each of three to five different influenza virus strains.

25. The nucleic acid construct of claim 24, wherein the coding sequence encodes the HA, fragment or variant of each of three or four non-influenza virus strains.

26. The nucleic acid construct of any of claims 21 to 25, wherein the coding sequence encodes the HA, fragment or variant of an influenza virus strain.

27. A vector particle coated with the nucleic acid construct of any one of claims 21 to 26.

28. A vector particle coated with at least two different nucleic acid constructs of claim 21, wherein each of said constructs encodes the HA, fragment or variant of a different influenza virus strain.

29. The vector particle of claim 28, coated with three to five different said constructs.

30. The vector particle of claim 29, wherein three or four of the constructs encode the HA, fragment or variant of a different non-influenza virus strain.

31. The vector particle of any one of claims 28 to 30, which is coated with said construct encoding said HA, fragment or variant of a pandemic influenza virus strain.

32. The vector particle of any one of claims 27 to 31, further coated with a nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to the promoter, wherein the coding sequence encodes an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof both having adjuvant activity and having at least 80% amino acid homology to the subunit or fragment.

33. The vector particle of claim 32, wherein the promoter sequence is a chimeric promoter sequence comprising:

(a) hCMV immediate early promoter sequence;

34. The vector particle according to claim 32 or 33, wherein said ADP ribosylating bacterial toxin subunit is selected from the group consisting of cholera toxin subunit a, cholera toxin subunit B, escherichia coli heat-labile toxin subunit a, and escherichia coli heat-labile toxin subunit B.

35. The vector particle of any one of claims 32 to 34, wherein said nucleic acid construct comprises two coding sequences, each comprising a different said subunit, fragment or variant.

36. The vector particle of claim 35, wherein the two coding sequences encode cholera toxin subunit a and cholera toxin subunit B, respectively, or escherichia coli heat-labile toxin subunit a and escherichia coli heat-labile toxin subunit B, respectively.

37. The vector particle of any one of claims 32 to 36, wherein the nucleic acid construct further comprises:

(a) a non-translated leader sequence derived from an HBVpreS2 antigen sequence, an HBV e-antigen sequence, or an HSV-type 2gD antigen sequence, and operably linked to the chimeric promoter; and/or

38. The support particle of any one of claims 27 to 37, which is a gold particle.

39. A dose container for a particle-mediated delivery device comprising the coated particles of any one of claims 27 to 38.

40. A particle-mediated delivery device loaded with the coated particle of any one of claims 27 to 38.

41. The particle-mediated delivery device of claim 40 which is a needleless syringe.

42. A nucleic acid construct comprising a chimeric promoter sequence and a coding sequence operably linked to the chimeric promoter, wherein the coding sequence encodes an influenza virus antigen, an immunogenic fragment thereof, or an immunogenic variant of the antigen or fragment having at least 80% amino acid homology to the antigen or fragment, and the chimeric promoter sequence comprises:

(a) hCMV immediate early promoter sequence;

43. The nucleic acid construct of claim 42, wherein the encoded antigen is influenza virus Hemagglutinin (HA), an immunogenic fragment thereof, or an immunogenic variant thereof having at least 80% amino acid sequence homology to both.

44. The nucleic acid construct of claim 42, wherein the encoded antigen is influenza virus Neuraminidase (NA), M2, an immunogenic fragment of both, or an immunogenic variant having at least 80% amino acid sequence homology to any of said NA, M2 or fragment.

45. The nucleic acid construct of any of claims 42 to 44, wherein the influenza virus antigen, immunogenic fragment or variant is from an epidemic influenza virus strain.

46. The nucleic acid construct of any of claims 42 to 45, wherein the construct encodes more than one influenza virus antigen, immunogenic fragment or immunogenic variant.

47. The nucleic acid construct of claim 46, wherein the construct encodes an influenza virus epidemic antigen, an immunogenic fragment thereof, or an immunogenic variant of said antigen or fragment, and encodes one or more non-influenza virus antigens, one or more immunogenic fragments thereof, or one or more immunogenic variants of said one or more antigens or one or more fragments.

48. The nucleic acid construct of claim 46 or 47, wherein at least two of the encoded different antigens, fragments or variants are from the same influenza polypeptide of different influenza strains.

49. A nucleic acid construct comprising a chimeric promoter sequence and a coding sequence operably linked to the chimeric promoter sequence, wherein the coding sequence encodes an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof which has at least 80% amino acid homology to the subunit or fragment, and the chimeric promoter sequence comprises:

(a) hCMV immediate early promoter sequence;

50. The nucleic acid construct of claim 49, wherein said ADP ribosylating bacterial toxin subunit is selected from the group consisting of cholera toxin subunit A, cholera toxin subunit B, Escherichia coli heat-labile toxin subunit A, and Escherichia coli heat-labile toxin subunit B.

51. The nucleic acid construct of claim 49 or 50, wherein said nucleic acid construct comprises two coding sequences, each of said coding sequences comprising a different of said subunits, fragments or variants, and each of said coding sequences is operably linked to one of said chimeric promoters.

52. The nucleic acid construct of claim 51, wherein said two coding sequences encode cholera toxin subunit A and cholera toxin subunit B, respectively, or E.coli heat-labile toxin subunit A and E.coli heat-labile toxin subunit B, respectively.

53. The nucleic acid construct of claim 51 or 52, wherein the two coding sequences are in opposite orientations.

54. The nucleic acid construct of claim 51 or 52, wherein the two coding sequences are in the same orientation.

55. The nucleic acid construct of any one of claims 42 to 54, wherein the hCMV immediate early promoter sequence (a) comprises:

(ii) (ii) a functional variant of (i) having at least 80% nucleotide sequence homology to one or more sequences of (i); or

(iii) (iii) a functional fragment of (i) or (ii).

56. The nucleic acid construct of any of claims 42 to 55, wherein the exon sequences (b) comprise:

(i) nucleotide sequence SEQ ID No.2, nucleotide 1588 to 1718 of nucleotide sequence SEQ ID No.54, nucleotide 1684 to 1814 of SEQ ID No.61, nucleotide 2633 to 2763 of SEQ ID No.61, nucleotide 1687 to 1817 of SEQ ID No.62 and/or nucleotide 3309 to 3439 of SEQ ID No. 62;

(iii) (iii) a functional fragment of (i) or (ii).

57. The nucleic acid construct of any of claims 42 to 56, wherein the heterologous intron (c) comprises a sequence selected from the group consisting of SEQ ID NO: a rat insulin gene intron a sequence, a chicken keratin gene intron a sequence, a chicken heart actin gene intron a sequence, a functional fragment thereof, or a functional variant of any of the foregoing.

58. The nucleic acid construct of claim 57, wherein the rat insulin gene intron A sequence comprises:

(iii) (iii) a functional fragment of (i) or (ii).

59. The nucleic acid construct of any of claims 42 to 58, wherein the chimeric promoter sequence comprises:

(ii) (ii) a functional variant of (i) having at least 80% nucleotide sequence homology to (i); or

(iii) (iii) a functional fragment of (i) or (ii).

60. The nucleic acid construct of any one of claims 42 to 59, further comprising:

(a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence and operably linked to the chimeric promoter; and/or

(b) An enhancer sequence derived from the 3 'untranslated region (UTR) of the HBsAg sequence or the 3' UTR of the simian CMV immediate early gene sequence, operably linked to the chimeric promoter, wherein the enhancer sequence is located downstream of the cloning site.

61. The nucleic acid construct of claim 42, comprising:

(i) provided as SEQ ID NO: 14, or of the vector pppJV 7563, or

(ii) (ii) a sequence having at least 60% sequence identity to the sequence of (i);

and the sequence encoding the antigen, fragment or variant is provided in the sequence (i) or (ii) such that it is operably linked to the chimeric promoter.

62. The nucleic acid construct of claim 61, wherein the coding sequence encodes an immunogenic fragment of the HA antigen, an immunogenic variant thereof, or both.

63. The nucleic acid construct of claim 42, comprising a sequence provided as SEQ ID NO: 54, or a sequence having at least 60% sequence identity thereto.

64. The nucleic acid construct of claim 42, comprising a sequence provided as SEQ ID no: 59, or a sequence having at least 60% sequence identity thereto.

65. The nucleic acid construct of claim 49, comprising a nucleic acid sequence consisting of SEQ ID No: 61, or a sequence having at least 60% sequence identity thereto, provided in vector pppjv 2012.

66. The nucleic acid construct of claim 49, comprising a nucleic acid sequence consisting of SEQ ID No: 62, or a sequence having at least 60% sequence identity thereto.

67. A nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to the promoter, wherein the coding sequence encodes an influenza virus antigen, an immunogenic fragment thereof, or an immunogenic variant of the antigen or fragment having at least 80% amino acid homology to the antigen or fragment, and the construct further comprises:

(a) A non-translated leader sequence derived from an HBV preS2 antigen sequence, an HBV e-antigen sequence or an HSV-type 2gD antigen sequence, operably linked to the coding sequence and a promoter heterologous to the coding sequence; and/or

68. The nucleic acid construct of claim 67, wherein the encoded antigen is influenza virus Hemagglutinin (HA), an immunogenic fragment thereof, or an immunogenic variant thereof having at least 80% amino acid sequence homology to both.

69. The nucleic acid construct of claim 67, wherein the encoded antigen is influenza virus Neuraminidase (NA) or M2, an immunogenic fragment of both, or an immunogenic variant having at least 80% amino acid sequence homology to said NA, M2, or fragment.

70. The nucleic acid construct of any one of claims 67 to 69, wherein the encoded influenza virus antigen, immunogenic fragment thereof, or immunogenic variant of both is from an influenza strain.

71. The nucleic acid construct of any one of claims 67 to 70, wherein the construct encodes more than one influenza virus antigen, immunogenic fragment or immunogenic variant.

72. The nucleic acid construct of claim 71, wherein the construct encodes an antigen of an epidemic influenza virus strain, an immunogenic fragment thereof, or an immunogenic variant of said antigen or fragment, and encodes one or more antigens of a non-epidemic influenza virus strain, one or more immunogenic fragments thereof, or one or more immunogenic variants of one or more of said antigens or one or more fragments.

73. The nucleic acid construct of claim 71 or 72, wherein at least two of the different antigens, fragments or variants are from the same influenza polypeptide of different influenza strains.

74. A nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to the promoter, wherein the coding sequence encodes an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof both having adjuvant activity and at least 80% amino acid sequence homology to the subunit or fragment, and the construct further comprises:

75. The nucleic acid construct of claim 74, wherein said ADP ribosylating bacterial toxin subunit is selected from the group consisting of cholera toxin subunit A, cholera toxin subunit B, Escherichia coli heat-labile toxin subunit A, and Escherichia coli heat-labile toxin subunit B.

76. The nucleic acid construct of claim 74 or 75, wherein said nucleic acid construct comprises two coding sequences, each of said coding sequences comprising a different of said subunits, fragments or variants, and each of said coding sequences is linked to said chimeric promoter.

77. The nucleic acid construct of claim 76, wherein said two coding sequences encode cholera toxin subunit A and cholera toxin subunit B, respectively, or E.coli heat-labile toxin subunit A and E.coli heat-labile toxin subunit B, respectively.

78. The nucleic acid construct of claim 76 or 77, wherein the two coding sequences are in opposite orientations.

79. The nucleic acid construct of claim 76 or 77, wherein the two coding sequences are in the same orientation.

80. The nucleic acid construct of any one of claims 67 to 79, wherein said promoter is selected from the group consisting of said hCMV immediate early promoter sequence, a pseudorabies virus promoter sequence, and a Rous sarcoma virus promoter sequence.

81. The nucleic acid construct of any of claims 67 to 80, wherein the untranslated leader sequence comprises:

(i) nucleotide sequences SEQ ID No 5, SEQ ID No: 6. SEQ ID No: nucleotide 1864 to 1984 of 7 or SEQ ID No. 54;

(ii) (ii) a functional variant of (i) which has at least 80% nucleotide sequence homology with (i); or

(iii) (iii) a functional fragment of (i) or (ii).

82. The nucleic acid construct according to any of claims 67 to 81, wherein the enhancer sequence comprises:

(i) nucleotide sequence SEQ ID No: 8. SEQ ID No: 9. nucleotides 3699 to 4231 of SEQ ID No.54, SEQ ID No: nucleotides 3831 to 4363 of 61 and/or SEQ ID No: nucleotides 4507 to 5038 of 62;

(ii) (ii) a functional variant of (i) having at least 80% nucleotide sequence homology to one of said sequences (i); or

(iii) (iii) a functional fragment of (i) or (ii).

83. The nucleic acid construct of any one of claims 42 to 82, further comprising a polyadenylation sequence.

84. The nucleic acid construct of claim 83, wherein said polyadenylation sequence is a polyadenylation sequence of a gene selected from the group consisting of: rabbit β -globin gene, Human Papilloma Virus (HPV) early or late gene, HSV-2gB gene, simian CMV immediate early gene, and HSVgD late gene.

85. The nucleic acid construct of claim 83, wherein said polyadenylation sequence is selected from the group consisting of:

(i) nucleotide sequence SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID No: 54 from nucleotide 4243 to 4373 of SEQ ID No: nucleotides 906 to 1038 of 61, SEQ ID No: nucleotides 4375 to 4050 of 61 or SEQ id no: nucleotides 2463 to 2593 of 62;

(ii) (ii) a functional variant having at least 80% nucleotide sequence homology to one of said sequences (i); or

(iii) (iii) a functional fragment of (i) or (ii).

86. The nucleic acid construct of claim 42, comprising the sequence SEQ ID NO: 14.

87. the nucleic acid construct of any one of claims 42 to 86, further comprising a nucleotide sequence encoding a signal peptide operably linked to said sequence encoding said antigen, exotoxin subunit, fragment, or variant.

88. The nucleic acid construct of claim 87, wherein said signal peptide is selected from the group consisting of human tissue plasminogen activator signal peptide (hTPASP), aprotinin signal peptide, tobacco extensin signal peptide, and chicken lysozyme signal peptide.

89. The nucleic acid construct of any one of claims 42 to 88, which is a plasmid.

90. A population of nucleic acid constructs, wherein the population comprises at least two different constructs of any one of claims 42 to 89.

91. A population of nucleic acid constructs, wherein the population comprises at least two different constructs of any one of claims 42 to 48, 61 to 64 and 67 to 73 encoding different influenza virus antigens, immunogenic fragments thereof or immunogenic variants of said antigens or fragments.

92. The population of claim 91, which comprises at least three different constructs, each construct encoding a different influenza virus antigen, immunogenic fragment, or immunogenic variant.

93. The population of nucleic acid constructs of claim 91 or 92, wherein at least two of said different antigens are from the same influenza polypeptide of different influenza strains.

94. The population of nucleic acid constructs of any one of claims 91 to 93, wherein at least two of said different antigens, fragments or variants are from different influenza polypeptides of said same or different influenza strains.

95. A population of nucleic acid constructs according to any one of claims 90 to 94, said population comprising at least one construct according to any one of claims 49 to 54, 65, 66 and 74 to 79 encoding said ADP ribosylating bacterial toxin subunit, fragment or variant thereof.

96. A population of nucleic acid constructs, wherein:

(i) at least one construct encodes a subunit of an ADP ribosylating bacterial toxin, a fragment thereof having adjuvant activity, or a variant thereof both having adjuvant activity and having at least 80% amino acid homology to said subunit or fragment; and

(ii) at least one construct encodes a Herpes Simplex Virus (HSV) antigen;

wherein the sequence encoding the subunit, fragment or variant and/or HSV antigen is operably linked to a chimeric promoter comprising:

(a) hCMV immediate early promoter sequence;

97. A population of nucleic acid constructs, wherein:

(ii) At least one of the constructs encodes an HSV antigen,

wherein at least one of said constructs further comprises:

(a) a non-translated leader sequence derived from an HBV preS2 antigen sequence, HBV e-antigen sequence or HSV-type 2gD antigen sequence, operably linked to the coding sequence of the construct and a promoter heterologous to the coding sequence; and/or

(b) An enhancer sequence located 3 'of and operably linked to the coding sequence of the construct, wherein the enhancer sequence is derived from the 3' UTR of an HBsAg sequence or the 3 'UTR of a simian CMV immediate early gene sequence and the coding sequence is heterologous to the 3' enhancer sequence.

98. A population of nucleic acid constructs comprising:

(i) a first nucleic acid construct comprising a chimeric promoter sequence and a coding sequence operably linked to said chimeric promoter, wherein said coding sequence encodes an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof having adjuvant activity and at least 80% amino acid homology to said subunit or fragment, and said chimeric promoter sequence comprises:

(a) hCMV immediate early promoter sequence;

(ii) a second nucleic acid construct encoding at least one HSV antigen.

99. A population of nucleic acid constructs comprising:

(i) a first nucleic acid construct comprising a promoter sequence and a coding sequence operably linked to said promoter, wherein said coding sequence encodes an ADP ribosylating bacterial toxin subunit, a fragment thereof having adjuvant activity, or a variant thereof both having adjuvant activity and at least 80% amino acid homology to said subunit or fragment, and said construct further comprises:

(b) An enhancer sequence located 3 'of the coding sequence and operably linked thereto, wherein the enhancer sequence is derived from the 3' UTR of the HBsAg sequence or the 3 'UTR of the simian CMV immediate early gene sequence and the coding sequence is heterologous to the 3' enhancer sequence;

(ii) A second nucleic acid construct encoding at least one HSV antigen.

100. The population of nucleic acid constructs of claim 99, comprising:

(i) a first nucleic acid construct comprising a nucleic acid sequence consisting of SEQ ID NO: 61 or a sequence having at least 60% sequence identity thereto, of the vector pppjv 2012; and

(ii) a second nucleic acid construct encoding an HSV antigen.

101. A purified isolated chimeric promoter sequence, wherein the chimeric promoter sequence is as defined in any one of claims 42 and 55 to 57.

102. A coated particle comprising a vector particle coated with the nucleic acid construct of any one of claims 42 to 89 or a population of nucleic acid constructs of any one of claims 90 to 100.

103. The coated particle of claim 102, wherein the support particle is a gold particle.

104. A dose container for a particle-mediated delivery device comprising a coated particle as defined in claim 102 or 103.

105. A particle-mediated delivery device loaded with the coated particles as defined in claim 102 or 103.

106. The particle-mediated delivery device of claim 105, which is a needleless syringe.

107. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 42 to 89 or a population of nucleic acid constructs of any one of claims 90 to 100 and a pharmaceutically acceptable carrier or excipient.

108. A vaccine composition comprising the nucleic acid construct of any one of claims 42 to 89 or a population of nucleic acid constructs of any one of claims 90 to 100.

109. The vaccine composition of claim 108, which is a multivalent vaccine comprising at least two different constructs of any one of claims 42 to 48, 61 to 64 and 67 to 73 encoding different influenza virus antigens, immunogenic fragments thereof or immunogenic variants of both.

110. The vaccine composition of claim 109 which is a trivalent, tetravalent, or pentavalent influenza vaccine.

111. Use of a nucleic acid construct as defined in any one of claims 42 to 89, a population of nucleic acid constructs as defined in any one of claims 90 to 95 or a coated particle as defined in claim 102 or 103 in the preparation of a medicament for the prevention of influenza.

112. The use of claim 111, wherein the medicament is to be delivered by injection, transdermal particle delivery, inhalation, topical administration, oral administration, intranasal administration, or transmucosal administration.

113. The use of claim 111 or 112, wherein the medicament is to be delivered by needle-free injection.

114. A method of obtaining in vitro expression of an influenza polypeptide of interest in a mammalian cell, the method comprising transferring the nucleic acid construct of any one of claims 42 to 89, a population of nucleic acid constructs of any one of claims 90 to 95, or the coated particle of claim 102 or 103 into the cell.