HK1170496B - Lentiviral vectors pseudotyped with a sindbis virus envelope glycoprotein - Google Patents

Description

Lentiviral vectors pseudotyped with Sindbis virus envelope glycoprotein

Technical Field

The present patent application relates generally to target gene delivery and more specifically to the use of pseudotyped lentiviruses comprising an envelope that targets dendritic cells and can therefore be used for dendritic cell vaccination.

Background

Dendritic Cells (DCs) are the basic antigen presenting cells used to initiate and control immune responses. DCs can capture and process antigens, migrate from peripheral organs to lymphoid organs, and present antigens to resting T cells in a Major Histocompatibility Complex (MHC) restricted manner. These cells are derived from Bone Marrow (BM) and exhibit dendritic morphology and high mobility. The discovery of DCs as specialized Antigen Presenting Cells (APCs) has prompted attempts at DC-based immunization/vaccination strategies involving in vitro loading of DCs with specific antigens (Banchereau and Palucka, A.K.2005.Nat.Rev.Immunol.5: 296-306; Figdor et al 2004.nat. Med.10: 475-480). However, all of these attempts involve labor intensive preparation of patient specific treatments involving ex vivo loading of autologous DCs with specific antigens prior to their administration to patients.

An alternative strategy is to use recombinant virus-based vectors as a mechanism for delivering genes encoding a given antigen directly to a host cell. In this case, the expressed gene product provides a therapeutic benefit via the introduction of the desired adaptive immune response. However, implementing a safe and effective system faces many challenges. Some of these challenges include designing a vector that targets a desired set of host cells, providing a suitable delivery system, expressing the desired antigen that elicits an effective immune response, and consistently making a pharmaceutical composition of recombinant viral vector virus of sufficiently high potency that the virus can be widely used in a given population of human subjects. The latter is a particular challenge in developing laboratory scale systems as products that can be produced by the pharmaceutical industry.

In the laboratory, many lentiviral vectors were pseudotyped with VSV-G envelope proteins. This is widely used as a model system because VSV envelope proteins are able to target many cell types ("panophilic" envelopes), and production systems generally provide high titers.

The envelope glycoproteins of sindbis virus and other alphaviruses disclosed herein are incorporated into the lipid bilayer of the virion membrane. Typically, the viral membrane (envelope) contains multiple copies of a trimer of the two glycoprotein heterodimers E1 and E2 resulting from the cleavage of a single precursor protein. From its N-terminus to C-terminus, precursor proteins comprise E3, E2, 6K, and E1 proteins. The small E3 glycoprotein serves as a signal sequence for translocation of the E2 protein into the membrane and is cleaved from E2 by furin (furin) or some other Ca2+ dependent serine protease. The 6K protein serves as a signal sequence for translocation of the E1 protein into the membrane, and subsequent cleavage from the precursor protein.

Each of the E1 and E2 glycoproteins has a transmembrane region; e2 has a cytoplasmic domain of about 33 residues, whereas the cytoplasmic tail of E1 is extremely short (about 2 residues). Both E1 and E2 have palmitic acid attached within or near the transmembrane region.

Isolates of sindbis virus described in the art are believed to infect cells via interaction with Heparan Sulfate (HS). A lentivirus packaging system is described in WO2008/011636 in which the E3/E2 envelope fusion protein (known as SVgmu) contains a number of modifications in order to reduce binding of proteins to HS via DC-SIGN surface molecules, but to retain binding to and infect DCs. In WO2008/011636, cDNA of wild-type SVG was obtained from the laboratory of dr.j.h.strauss laboratory of california institute of technology and cloned by PCR into pcDNA3 vector (Invitrogen) to produce plasmid pSVG. A 10 residue tag sequence was inserted between amino acids 71 and 74 in the E2 protein by PCR mutagenesis to disrupt the HS binding site. An additional deletion was introduced into the E3 glycoprotein of SVG to remove amino acids 61-64. This modified SVG is referred to as SVGMu (SEQ ID NO: 11 of WO 2008/011636). The cDNA of SVGMu was cloned downstream of the CMV promoter in pcDNA3 vector (designated pSVGmu, SEQ ID NO: 3 of WO 2008/011636).

Some embodiments of the invention

Although SVGmu pseudotyped virions are able to selectively transduce cells expressing DC-SIGN antigen, several aspects of the system make it unsuitable for therapeutic use. For example, the E3/E2 fusion protein displays antigenic epitopes of influenza hemagglutinin, integration of the viral genome into the host chromosome can activate deleterious host genes, and the inventors have found that the titer of the virus is low compared to the titer of particles pseudotyped with the VSV-G envelope. Remarkably, sindbis virus strains (such as SVGmu) with mutations that prevent the correct processing of E3 from the E2 glycoprotein (the so-called "pE 2 mutant") grow poorly in permissive cell lines and are severely attenuated in mouse pathogenicity.

The alignment of the SVGmu E3-E2 protein used to pseudotype lentiviral vectors in WO2008/011636 with three exemplary E protein variants of the invention is shown in figure 1. The numbers indicated in FIG. 1 are with reference to the HR strain of the Sindbis virus envelope protein. Unless otherwise indicated, the numbering used herein is with reference to such strains of sindbis virus. The inventors have modified the SVGmu protein to provide an improved lentivirus production system. The virus particles of the present invention are prepared at significantly higher titers than those using SVGmu and also stimulate a stronger immune response. Furthermore, the titer of pseudotyped lentiviral particles was increased and infectivity improved while selectivity for DC was retained.

In one embodiment, the invention provides a retroviral (such as lentiviral) vector particle comprising: (a) an envelope comprising the amino acid sequence of SEQ ID NO:1, wherein 160X is absent or is a non-acidic amino acid, or SEQ ID NO:1, which is capable of infecting a dendritic cell; wherein the E2 glycoprotein or variant thereof is not fused to E3; and (b) a lentiviral genome comprising a sequence of interest.

Variants may be capable of binding to DC-SIGN. It is also preferred that the variant has reduced binding to heparan sulphate compared to a reference protein from a strain of HR. The E2 protein of the HR strain is shown as SEQ ID NO: 18.

preferably, 160X is one of a small amino acid or an aliphatic amino acid, including glycine, alanine, valine, leucine, or isoleucine. In one aspect, 160X is absent or is glycine, valine, leucine, or isoleucine. In one embodiment, X is glycine.

SEQ ID NO:1 is defined as a variant comprising an amino acid sequence identical to SEQ ID NO:1 or at least 82%, 85%, 87%, 90%, 92%, 95%, or 98% sequence identity, wherein 160X is retained and as defined above. A variant may have one or more of lysine and arginine in the region spanning residues 50 to 180, independently deleted or substituted with a non-basic amino acid. In one embodiment, the non-basic amino acid is glutamic acid or aspartic acid.

In one aspect, residue X is selected from deletion, glycine, valine, leucine or isoleucine, and one or more of lysine and arginine in the region spanning residues 50 to 180 is independently deleted or substituted with a non-basic amino acid.

In another aspect, X is selected from deletion, glycine, alanine, valine, leucine or isoleucine, and one or more of lysine and arginine in the region spanning residues 50 to 180 (preferably including at least position 159) are independently deleted or substituted with glutamic acid or aspartic acid.

Candidate positively charged amino acids that may be substituted or deleted include lysines at residues 63, 70, 76, 84, 97, 104, 129, 131, 133, 139, 148, 149 and 159 and arginines at residues 65, 92, 128, 137, 157, 170 and 172 (numbered as SEQ ID NO: 1). When substituted, the substitutions may be independently selected from glutamic acid or aspartic acid.

In particular embodiments, one or more of lysine residues 70, 76, and 159 are deleted or substituted. When substituted, the substitutions may be independently selected from glutamic acid or aspartic acid.

Since sindbis virus has an RNA genome, the sequences that are identical to SEQ ID NO:1, for SEQ ID NO: 1-including substitutions, insertions or deletions-in addition to those mentioned above. For example, there is SEQ ID NO:1, wherein position 3 is T or V, 23 is V, 209 is R, 264 is G and 393 is H. The sequence can be shown for SEQ ID NO:1 making one or more of these changes or other changes, provided that the variant retains the ability to infect DCs.

In one embodiment, the variant is represented in SEQ ID NO:1 does not contain any insertions in the region between residues 70 and 76. In this embodiment, SEQ ID NO:1 may be unchanged or may comprise one or two substitutions that do not affect the ability of the variant to infect DCs.

In some cases, the E2 protein is first expressed as a multimeric protein fused to at least E3 or to a leader sequence. In certain embodiments, E2 is expressed as part of the E3-E2-6K-E1 polyprotein. Sindbis virus naturally expresses E2 as part of a polyprotein, and the junction regions of E3/E2, E2/6K, and 6K/E1 have sequences recognized and cleaved by endopeptidases. The sequence of the E3/E2 polyprotein is SEQ ID NO: 20. typically, the E3/E2 junction is cleaved by furin or a furin-like serine endopeptidase between residues 65 and 66 of the E3/E2 polyprotein. Furin is specific for a pair of arginine residues separated by two amino acids. To maintain cleavage by furin E3/E2, residues 62-66 (RSKRS; SEQ ID NO: 26) should maintain two arginine residues and a serine residue separated by two amino acids.

In one embodiment of the invention, the polymeric protein comprises a nucleotide sequence corresponding to SEQ ID NO: 20 or a variant thereof which hybridizes to residues 1-65 of SEQ ID NO: 20 or at least 82%, 85%, 87%, 90%, 92%, 95% or 98% sequence identity, wherein residues 62-65 are RSKR (SEQ ID NO: 27) and the variant is capable of being incorporated into a pseudotyped viral envelope. Preferably, the E2 portion of the polymeric protein is any embodiment as defined herein above.

Alternatively, a different cleavage sequence may be used in place of the E3/E2 furin cleavage sequence or any other cleavage sequence. Recognition and cleavage sites for endopeptidases can be incorporated, including without limitation: aspartic endopeptidases (e.g., cathepsin D, chymosin, HIV protease), cysteine endopeptidases (bromelain, papain, calpain), metalloendopeptidases (e.g., collagenase, thermolysin), serine endopeptidases (e.g., chymotrypsin, factor IXa, factor X, thrombin, trypsin), streptokinases. The recognition and cleavage site sequences of these enzymes are well known.

Where such a cleavage site is used, it may be introduced into an E3 polymeric protein, said E3 polymeric protein comprising at least the amino acid sequence of SEQ ID NO: 20 (such as residues 1-61 or 1-62) or a variant thereof which is identical to SEQ ID NO: 20, or at least 82%, 85%, 87%, 90%, 92%, 95%, or 98%, and the E3 polymeric protein is fused directly at the C-terminus to the cleavage sequence, which in turn is fused to the E2 portion of the polymeric protein. Preferably, the E2 portion of the polymeric protein is any embodiment as defined herein above.

The E2 protein sequence of the present invention includes SEQ ID NO:1, wherein 160X is as defined in the following table and the protein is as set forth in SEQ ID NO:1 except for the following residues 70, 76 and 159 and 160 as follows:

optionally, each of the above sequences may comprise a sequence selected from the group consisting of SEQ ID NOs: 1, but with NO change yet to SEQ ID NO:1, or may have one or two substitutions that do not affect the ability of the variant to infect DCs, but do not alter the number of amino acids in this region.

Other E2 protein sequences of the invention include SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 or SEQ ID NO: 15, wherein residue 160 is Ala, lle, Leu, or Val instead of Gly. Such variants may also comprise NO change yet to SEQ ID NO:1, or may have one or two substitutions that do not affect the ability of the variant to infect DCs, but do not alter the number of amino acids in this region.

Optionally, SEQ ID NO: 3-15 or other variants may be varied at other positions to provide sequences identical to SEQ ID NOs: 1E 2 sequence having at least 80% sequence identity. "at least 80% sequence identity" includes at least 82%, 85%, 87%, 90%, 92%, 95%, or 98% sequence identity.

In the above embodiments, the nucleic acid sequence corresponding to SEQ ID NO: the first E2 residue at position 66 of 20 may be Ser.

As indicated above, the E2 sequence of the invention, including the SEQ ID NO: 3-15 and variants thereof, may be expressed from a polyprotein comprising at least the sequences E3/E2, and preferably the sequences sindbis E3/E2/6K/E1 polyprotein. The E3 polyprotein sequence in each embodiment can be SEQ ID NO: 20 or any variant thereof, including variants having a non-natural cleavage site.

Disclosure of Invention

The present patent application relates to pseudotyped lentiviral vectors comprising a genome having a sequence of interest and an envelope comprising glycoproteins of an arbovirus. The arbovirus glycoprotein may be from Sindbis virus, dengue virus and Venezuelan equine encephalitis virus. Specifically, when the glycoprotein is the E2 protein from sindbis virus, the amino acid sequence is identical to SEQ ID NO:1 in contrast, the E2 protein has at least one amino acid change at residue 160. The amino acid change may be a deletion or an amino acid other than glutamic acid. Alternatively, the E2 glycoprotein may be a variant protein that hybridizes with SEQ ID NO:1 has at least 80% sequence identity and also has an alteration of residue 160 which is a deletion or an amino acid other than glutamic acid. The glycoprotein facilitates infection of dendritic cells. In all cases, the E2 glycoprotein is not part of a fusion protein with sindbis virus E3. In some embodiments, the E2 glycoprotein or variant binds to DC-SIGN. The lentiviral vector also comprises a lentiviral genome comprising the sequence of interest.

In certain embodiments, residue 160 is absent or is glycine, alanine, valine, leucine, or isoleucine. In one embodiment, residue 160 is glycine. In addition, other alterations of the E2 glycoprotein may occur in combination with the mentioned alterations of residue 160. One such change is an amino acid change that reduces the net positive charge of E2. One way to reduce the net positive charge is to change a lysine to a non-basic amino acid. In particular embodiments, one or more of lysine 70, lysine 76, or lysine 159 is altered. In certain embodiments, one or more of these lysines are changed to glutamic acid or aspartic acid. In particular embodiments, the E2 glycoprotein is SEQ ID NO: 3-16. Examples of combinations of alterations include, without limitation, altering glutamic acid at position 160 to a non-basic residue, and altering one or more of lysine 70, lysine 76, or lysine 159 to a non-basic residue. Other changes may also be made, in combination with the change at residue 160 and optionally any of the other changes disclosed herein. For example, any of the previously mentioned E2 glycoproteins may optionally comprise one or more further substitutions, insertions or deletions. As a specific example, the protein cleavage site between E2 and E3 may be the native sequence or an altered sequence that is cleaved by a different endopeptidase. In other embodiments that may be combined with any of the above, the nucleic acid sequence of SEQ ID NO:1, or may have one or two amino acid substitutions that do not affect the ability of the variant to infect DCs.

In certain embodiments, the lentiviral vector genome of any of the above virions comprises a sequence of interest encoding a tumor-specific antigen or a virus-derived antigen, such as an HIV or SIV antigen. In some embodiments, any of the carrier particles are prepared at a titer of at least 105/mL IU.

In another aspect, there is provided a lentiviral vector packaging system for making a pseudotyped lentiviral vector particle, comprising: a first nucleic acid molecule encoding the polypeptide of SEQ ID NO:1, wherein residue 160 is absent or is an amino acid other than glutamic acid, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, said variant having the amino acid sequence of SEQ ID NO:1 and wherein residue 160 is absent or is an amino acid other than glutamic acid, and wherein the amino acid residue is at least 80% sequence identity; a second nucleic acid molecule encoding gag and pol proteins; a third nucleic acid molecule encoding rev; and a lentiviral vector genome comprising a sequence of interest. The E2 glycoprotein or variant of the packaging system has an amino acid sequence as defined in any of the embodiments above. In certain embodiments, the pol protein has a non-functional integrase. In a particular embodiment, the non-functional integrase has the D64V mutation. In some embodiments, the second nucleic acid molecule is a non-integrated lentiviral genome. In particular embodiments, att sites are mutated or deleted or PPT sites are mutated or deleted or both. The non-integrated lentiviral genome can be used in combination with a non-functional integrase and in combination with any one of the E2 proteins or variants. Preferably, the lentiviral vector particles are prepared to have a titer of at least 105 IU/mL. In some cases, the cell is transfected with the first nucleic acid molecule and the fourth nucleic acid molecule described above. The cell may already comprise the stably transformed second nucleic acid molecule and the third nucleic acid molecule.

There is provided an isolated nucleic acid molecule encoding a glycoprotein as described above, or an E3/E2 glycoprotein optionally in the form of a sindbis E3/E2/6K/E1 polyprotein, or wherein the E3 sequence corresponds to SEQ ID NO: 20, or a variant thereof which is identical to the amino acid sequence of SEQ ID NO: 20, wherein residues 62-65 are RSKR (SEQ ID NO: 27) and the variant is capable of being incorporated into a pseudotyped viral envelope, optionally further wherein residue 1 of the E2 polyprotein is Ser. The E2 glycoprotein can be any of the variants described above, including combinations of alterations. Furthermore, an expression vector comprising the nucleic acid molecule is provided, as well as a host cell comprising the expression vector.

There is provided a method of preparing lentiviral vector particles having any of the variants or combinations of the above, comprising: expressing in a cell a first nucleic acid molecule encoding the polypeptide of SEQ ID NO:1, wherein residue 160 is other than glutamic acid, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, which variant differs from SEQ ID NO:1 has at least 80% sequence identity and wherein residue 160 is absent or is an amino acid other than glutamic acid, and; (ii) expressing a second nucleic acid molecule, wherein the second nucleic acid molecule can be transcribed and the transcript can be assembled into a pseudotyped lentiviral vector particle.

Any lentiviral vector particle may be used in a method of treatment of a human or animal subject. The treatment may be a vaccine for immunization, wherein the vaccine is prophylactic or therapeutic. The vaccine comprises lentiviral vector particles and a pharmaceutically acceptable excipient. Alternatively, the lentiviral vector particles can be administered to the cell in vitro, comprising mixing the cell with any of the lentiviral vector particles described above.

These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. Further, U.S. application No. 61/228,491, filed on 24/7/2009, is incorporated by reference herein in its entirety for all purposes.

Drawings

FIG. 1 is an alignment of the envelope proteins of four Sindbis virus envelopes (SVGMu, SIN-Var1, SIN-Var2, and SIN-Var 3). The alignment is shown relative to sindbis envelope SVGmu described previously. The main differences from SVgmu include regeneration of the furin-like protease cleavage site (RSKR; SEQ ID NO: 27) between E3 and E2, removal of the HA epitope tag, and a series of lysine substitutions to reduce heparin binding.

FIG. 2 is a schematic of an exemplary vector for use in packaging viral particles.

FIG. 3 presents a graph of crude supernatant titers of lentiviral vector particle formulations, where the vector genome is pseudotyped by three different Sindbis virus envelope proteins (SVGMu and SIN-HR). Viral supernatants were generated by transient transfection using standard methods and were collected 48 hours post-transfection. Titers were determined on 293T cells expressing human DC-SIGN (293T-DC-SIGN). Titers are expressed as the number of GFP expression units per ml of supernatant and are the average of three independent transfections. Error bars represent standard deviation from the mean.

FIGS. 4A, 4B and 4C are graphs showing the immunological response of T cells in mice after administration of pseudotyped lentiviral vector particles. (A) C57BL/6 mice were immunized subcutaneously with one of two doses of an OVA-encoding integration-deficient lentiviral vector (designated as ng p 24). On day 9, the number and function of OVA 257-specific CD8T cells in the spleen were determined by MHC-I/peptide multimer and intracellular cytokine staining. (B) C57BL/6 mice were immunized subcutaneously with a range of doses of integration-deficient lentiviral vectors encoding OVA. On day 11, the percentage of OVA 257-specific CD8T cells in the spleen was determined by MHC-I/peptide multimer staining. (C) C57BL/6 mice were immunized subcutaneously with a range of doses of integration-deficient lentiviral vectors encoding OVA. On day 9, the percentage of OVA 257-specific CD8T cells in the spleen was determined by intracellular cytokine staining.

Fig. 5A presents a diagram of an exemplary lentivirus genome. Figure 5B presents the sequence of the U3 region of the three vector constructs. (A) The elements contained in all lentiviral vectors are shown on top within the exemplary vector genome. Promoters used include the human ubiquitin-C promoter (UbiC), the cytomegalovirus early transient promoter (CMV), or the Rous Sarcoma Virus (RSV) promoter. In addition to the standard SIN U3 region, a series of extended deletions were shown. Sequence alignment of the U3 region from all 3 vectors is shown in (B). The sequence shown includes a deletion of the polypurine channel (PPT) in construct 704, and the extended U3 deletion present in the 703 and 704 constructs.

Fig. 6A and 6B show GFP expression from lentiviral vectors following transduction of 293T cells. In fig. 6A, GFP is operably linked to the UbiC promoter, and in fig. 6B, GFP is operably linked to the CMV promoter. GFP expression levels were determined by integrase-deficient lentiviral vectors 48 hours after transduction of 293T cells expressing DC-SIGN. Measuring GFP expression in the transduced cells by standard flow cytometry methods; a total of 50,000 events were collected from each transduced cell pool to determine the average expression level.

Fig. 7 shows the number of GFP-positive cells during 5 passages. Cells were transduced with different vector preparations and passaged every 72 hours. Relative GFP titers were determined in 293T cell cultures transduced with different NILV constructs. The vectors were packaged using either wild-type integrase (IN +) or the D64V mutant integrase (IN-) and used to transduce 293 cells expressing DC-SIGN. The transduced cell cultures were then passaged every 72 hours for 15 days. At each passage, the number of GFP + cells in culture was determined using standard flow cytometry methods. The loss of GFP expression with passage indicates the loss of vector episome over time.

FIG. 8 shows CD8T cell responses following administration of either an integrating (Intwt) or non-integrating (IntD64V) lentiviral vector. Mice were immunized subcutaneously with C57BL/6 using a 2.5 x 1010 genome encoding either an integrative (Intwt) or non-integrative (IntD64V) lentiviral vector encoding Gag antigen from Simian Immunodeficiency Virus (SIV). On day 10, the number of antigen-specific T cells in the spleen and their cytokine secretion profile were determined by intracellular cytokine staining.

Figure 9 presents graphs showing tumor size (left panel) and percent survival (right panel) for mice receiving vehicle alone or virions encoding tumor antigens. BALB/c mice were injected subcutaneously with 2X 104 CT26 colon carcinoma cells. One day later, mice were treated subcutaneously with vehicle or 3.2 μ g (p24 capsid) of a DC-targeting non-integrating lentiviral vector (DC-NILV) encoding an AH1A5 peptide (SPSYAYHQF; SEQ ID NO: 25), a modified CT26 CD8T cellular epitope. Initial tumor growth and long-term survival of vaccinated mice compared to control mice are depicted.

Detailed Description

The present disclosure provides methods and compositions for targeting Dendritic Cells (DCs) by using lentiviral vector particles (e.g., virions, lentiviral particles) to deliver a sequence of interest to the DCs. The lentiviral vector particle comprises a variant of the envelope glycoprotein derived from sindbis virus E2 and a genome comprising a sequence of interest, and optionally other components. Glycoprotein variants showed reduced binding to heparan sulfate compared to HR, a reference sindbis virus strain. The envelope glycoprotein facilitates infection of dendritic cells by lentiviral vector particles. As used herein, "promoting" infection is the same as promoting transduction and refers to the role of the envelope glycoprotein, alone or in combination with other molecules, in promoting or enhancing receptor-mediated entry of pseudotyped retroviruses or lentiviral particles into target cells.

Typically, lentiviral vector particles are produced by a cell line containing one or more plasmid vectors and/or integrators which together encode the components necessary to produce a functional vector particle. These lentiviral vector particles are generally not replication competent, that is, they are only capable of a single round of infection. Most often, multiple plasmid vectors or independent expression cassettes stably integrated into the producer cell chromosome are used to separate the various genetic components that produce the lentiviral vector particles, however, a single plasmid vector with all lentiviral components can be used. In one example, the packaging cell line is transfected with the following plasmids: one or more plasmids containing a viral vector genome comprising LTRs (a cis-acting packaging sequence) and a target sequence; at least one plasmid encoding viral enzymatic and structural components (e.g., gag and pol); and at least one plasmid encoding an arbovirus envelope glycoprotein. The virion buds through the cell membrane and comprises a nucleus that typically contains two RNA genomes containing the sequence of interest and an arbovirus envelope glycoprotein that targets dendritic cells. When the arbovirus glycoprotein is sindbis virus E2 glycoprotein, the glycoprotein is engineered to reduce binding to heparan sulfate compared to the reference strain HR. This typically involves at least one amino acid change as compared to the HR E2 glycoprotein sequence.

Without wishing to be bound by theory, it is believed that binding of the virion to the cell surface induces endocytosis, allowing the virus to enter the endosome, triggering membrane fusion and allowing the viral nucleus to enter the cytosol. For certain embodiments that utilize integrating lentiviral vector particles, the viral genome integrates into the target cell genome after reverse transcription and migration of the product to the nucleus, thereby incorporating the sequence of interest into the genome of the target cell. However, to reduce the chance of insertional mutagenesis of a given antigen and to facilitate its transient expression, other embodiments utilize non-integrating lentiviral vector particles that do not integrate into the target cell genome, but instead express the sequence of interest from episomes. Either way, the infected DCs then express the target sequence, e.g., antigen, stimulatory molecule. The antigen can then be processed by dendritic cells and presented to T and B cells, generating an antigen-specific immune response. The specific pathway described above is not required as long as the dendritic cells are capable of stimulating an antigen-specific immune response.

The viral particles can be administered to a subject in order to provide a prophylactic or therapeutic effect. The product of the target sequence is typically a disease-causing factor or an antigen of a diseased cell (e.g., a tumor cell). Following infection of the dendritic cells and expression of the product, an immune response to the product is generated. The immune response may be a humoral immune response or a cellular immune response or both.

A. Viral vector envelopes

Arthropod-borne viruses (arboviruses) are viruses that are transmitted by an infected arthropod vector, such as a mosquito, to a host, such as a human, horse, or bird. Arboviruses are further divided into virus subfamilies including alphaviruses and flaviviruses, which have a single-stranded RNA genome with positive polarity and an envelope containing glycoproteins. For example, dengue, yellow fever and West Nile virus (West Nile virus) belong to the flaviviridae family, while sindbis, Semliki Forest virus (Semliki Forest virus) and venezuelan equine encephalitis virus are members of the alphavirus family (Wang et al j. virol.66, 4992 (1992)). The envelope of sindbis virus comprises two transmembrane glycoproteins (Mukhopadhyay et al Nature rev. microbio.3, 13 (2005)): e1, which is believed to contribute to fusion; and E2, which is believed to contribute to cell binding. Sindbis virus envelope glycoproteins are known to pseudotype other retroviruses, including oncogenic retroviruses and lentiviruses.

As described above, the arbovirus envelope glycoprotein can be used to pseudotype a lentiviral-based vector genome. A "pseudotyped" lentivirus is a lentivirus particle having one or more envelope glycoproteins encoded by a virus distinct from the lentivirus genome. The envelope glycoprotein may be modified, mutated or engineered as described herein.

The envelopes of sindbis virus and other alphaviruses are incorporated into the lipid bilayer of the virion membrane and typically comprise multiple copies of the two glycoproteins E1 and E2. Each glycoprotein has a transmembrane region; e2 has a cytoplasmic domain of about 33 residues, whereas the cytoplasmic tail of E1 is extremely short (about 2 residues). E1 and E2 have palmitic acid attached within or near the transmembrane region. E2 was initially synthesized as a precursor protein that was cleaved by furin or other Ca2+ -dependent serine proteases into E2 and a small glycoprotein called E3. Located between the sequences encoding E2 and E1 is a sequence encoding a protein called 6K. E3 and 6K are signal sequences used to translocate the E2 and E1 glycoproteins, respectively, into the membrane. In the sindbis virus genome, the coding region for sindbis envelope proteins contains sequences encoding E3, E2, 6K and E1. As used herein, the "envelope" of an arbovirus virus comprises at least E2, and may also comprise E1, 6K, and E3. An exemplary sequence of the envelope glycoprotein of sindbis virus (strain HR) is presented as SEQ ID No. 17. The sequences of envelope glycoproteins of other arboviruses can be found, for example, in gene banks. For example, the sequence encoding the dengue virus glycoprotein can be found at accession number GQ252677 (especially in the genbank) and in the virus variation database at NCBI (the genbank accession number and virus variation database are incorporated by reference to illustrate the sequence of the envelope glycoprotein), and the sequence encoding the venezuelan equine encephalitis virus envelope glycoprotein can be found at accession number NP 040824 (which is incorporated by reference to illustrate the sequence of the envelope glycoprotein).

Although, to date, cellular receptors on dendritic cells have not been finally identified, particularly for alphavirus and sindbis virus, one receptor appears to be DC-SIGN (Klimstra et al, J Virol 77: 12022, 2003). The terms "link," "bind," "target," and the like are used interchangeably and are not intended to indicate a mechanism of interaction between sindbis virus envelope glycoproteins and cellular components. DC-SIGN (non-integrating protein that captures dendritic cell-specific ICAM-3 (intracellular adhesion molecule 3); also known as CD209) is a C-type lectin-like receptor that rapidly binds and endocytoses substances (Geijtenbeek, T.B. et al, Annu. Rev. Immunol.22: 33-54, 2004). E2 appears to target the virus to dendritic cells via DC-SIGN. As shown herein, cells expressing DC-SIGN are transduced by viral vector particles pseudotyped with sindbis virus E2 better (at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold) than isogenic cells that do not express DC-SIGN. The mechanism of how the E2 glycoprotein facilitates viral infection appears to involve DC-SIGN, perhaps via direct binding to DC-SIGN, or causing a change in configuration or some other mechanism. Regardless of the actual mechanism, targeting by E2 is preferred for cells expressing DC-SIGN (i.e., dendritic cells).

Sindbis virus also appears to bind to cells via heparan sulfate (Klimstra et al, J Virol 72: 7357, 1998; Burmes and Griffin, J Virol 72: 7349, 1998). Because heparan sulfate and other cell surface glycosaminoglycans are found on the surface of most cell types, it is desirable to reduce the interaction between heparan sulfate and sindbis envelope glycoprotein. This can be accomplished by reducing the binding of sindbis viral envelope to heparan sulfate or increasing the binding of sindbis viral envelope to dendritic cells (e.g., increasing avidity) or both. Thus, non-specific binding to other molecules that may be expressed by other cell types and may occur even where the envelope is specific for DC-SIGN is reduced, and the improved specificity may be used to avoid undesirable side effects such as side effects that may reduce a desired immune response or side effects associated with off-target transduction of other cell types. Alternatively or in addition to the advantages of relatively specific transduction of cells expressing DC-SIGN, virions pseudotyped with the Sindbis virus envelope E2 glycoprotein may offer other advantages over virions pseudotyped with glycoproteins such as VSVG. Examples of such advantages include reduced complement-mediated lysis and/or reduced neuronal cell targeting, both of which are believed to be associated with administration of VSV-G pseudotyped virions.

In various examples, lentiviral vector particles specifically bind to cells expressing DC-SIGN and have reduced or eliminated binding to heparan sulfate. That is, the sindbis virus envelope E2 glycoprotein can be modified to direct the virus preferentially to dendritic cells expressing DC-SIGN over other cell types. Based on information obtained from molecular modeling in structural and other studies, variant sequences of envelope proteins (particularly the E2 and E1 glycoproteins) were designed and generated such that the glycoproteins retained their function as envelope proteins, but had the desired binding specificity, avidity, or level of binding. Candidate variant sequences for various glycoproteins can be generated and determined using the methods described below or other methods known in the art to identify envelope glycoproteins having the most desirable characteristics.

Certain variant sequences of sindbis E2 are identical to SEQ ID NO:1 compared to at least one amino acid change at residue 160. Residue 160 is deleted or changed to an amino acid other than glutamic acid. An alteration is most typically a substitution of at least one amino acid, but may alternatively be an addition or deletion of one or more amino acids. Preferably, any additional amino acids are few in number and do not include epitopes (e.g., hemagglutinin tag sequences) that may compromise safety. When two or more changes are present, they may be of the same type (e.g., substitution) or of different types (e.g., substitution and deletion). Multiple changes may be interspersed or contiguously located within the protein sequence.

In the first instance, the variant sequence comprises at least one amino acid change in the region of about residue 50 to about residue 180. Amino acids in this region are involved in binding to heparan sulfate. By reducing the net positive charge of E2, the electrostatic interaction with heparan sulfate can be reduced, resulting in reduced binding to heparan sulfate. Candidate positively charged amino acids within this region include lysine at residues 63, 70, 76, 84, 97, 104, 129, 131, 133, 139, 148, 149, 159 and arginine at residues 65, 92, 128, 137, 157, 170, 172 (Bear et al, Virology 347: 183-190, 2006). At least some of these amino acids are directly involved in the binding of E2 to heparan sulfate. The net positive charge may be reduced by deleting lysine or arginine or substituting lysine or arginine with a neutral or negatively charged amino acid. For example, one or more of these lysines and arginines may be replaced by glutamic acid or aspartic acid. Certain embodiments have a substitution to at least one of lysine 70, 76, or 159. In the case where E2 is expressed as a polyprotein with E3, the lysine located adjacent to the native E3/E2 cleavage site is retained-that is, the recognition sequence and cleavage site are not altered. Alternatively, the native endopeptidase cleavage site sequence is replaced by a recognition sequence for a different endopeptidase.

Certain variants of E2 were also modified in a manner that positively affected dendritic cell binding. The glutamate alteration seen at residue 160 in the reference HR can enhance binding to dendritic cells (see Gardner et al, J Virol 74, 11849, 2000, incorporated herein in its entirety). In certain variants, alterations such as deletion of residue 160 or substitution of residue 160 are found. In certain variants, uncharged amino acids are substituted with Glu, and in other variants, non-acidic amino acids are substituted with Glu. Typically, Glu160 is substituted with one of a small amino acid or an aliphatic amino acid, including glycine, alanine, valine, leucine, or isoleucine.

Other variants comprise two or more amino acid changes. Typically in these variants, one of the changes is Glu160 and the remaining changes are changes to one or more of lysine and arginine in a region spanning residues from about 50 to about 180. Certain variants comprise an alteration or deletion of Glu160 to a non-acidic residue, and one or more alterations of lysine 70, lysine 76, or lysine 159 by a non-basic amino acid. Some particular variants comprise Glu160 to Gly, Lys 70 to Glu, and Lys159 to Glu; glu160 to Gly, Lys 70, 76, and 159 to Glu; deletion of Glu160 and Lys 70 and 159 to Glu; and the absence of Glu160 and Lys 70, 76 and 159 to Glu.

In some cases, the E2 protein is first expressed as a multimeric protein fused to at least E3 or to a leader sequence. Regardless of whether the leader sequence is E3 or another sequence, E2 in the viral envelope should not have E3 or other leader sequences. In other words, E2 is preferably not an E3/E2 fusion protein (e.g., the E3/E2 fusion protein known as SVGMu). In certain embodiments, E2 is expressed as part of the E3-E2-6K-E1 polyprotein. Sindbis virus naturally expresses E2 as part of a polyprotein, and the junction regions of E3/E2, E2/6K, and 6K/E1 have sequences recognized and cleaved by endopeptidases. Typically, the E3/E2 junction is cleaved between residues 65 and 66 by furin or a furin-like serine endopeptidase. Furin is specific for a pair of arginine residues separated by two amino acids. To maintain cleavage by furin E3/E2, residues 62-66 (RSKRS; SEQ ID NO: 26) should maintain two arginine residues and a serine residue separated by two amino acids. Alternatively, a different cleavage sequence may be used in place of the E3/E2 furin cleavage sequence or any other cleavage sequence. Recognition and cleavage sites for endopeptidases can be incorporated, including but not limited to: aspartic endopeptidases (e.g., cathepsin D, chymosin, HIV protease), cysteine endopeptidases (bromelain, papain, calpain), metalloendopeptidases (e.g., collagenase, thermolysin), serine endopeptidases (e.g., chymotrypsin, factor IXa, factor X, thrombin, trypsin), streptokinases. The recognition and cleavage site sequences of these enzymes are well known.

The amino acid in E2, which differs from those already mentioned, can also be changed. Typically, a variant E2 sequence will have at least 80% sequence amino acid identity to the reference E2 sequence, or it may have at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. The variant glycoprotein should exhibit a biological function, such as the ability to facilitate infection of dendritic cells by a virion having an envelope comprising E2. Experiments have identified regions of the envelope glycoprotein that appear to play an important role in viral assembly, attachment to the cell surface, and various aspects of infection. The following information can be used as a guide when preparing variants. The cytoplasmic tail of E2-approximately residues 408 to 415-is important for virus assembly (West et al J Virol 80: 4458-4468, 2006; incorporated in its entirety). Other regions are involved in the formation of secondary structures (approximately residues 33-53); and involved in transport and protein stability (about residues 86-119) (Navaramajah et al, JVirol 363: 124-. The variant may retain the hydrophobic character of the region spanning approximately residues 370-380 of the membrane. Variants may retain one or both of the N-linked glycosylation site residues NIT (residue 196-198) and NFT (residue 318-320) and may retain one or more sites of palmitoylation (C-396, C416 and C417) (Strauss and Strauss Microbiol Rev 58, 491-562, 1994; incorporated at pages 499-509). On the other hand, many regions of E2 may be changed without the occurrence of a harmful event. For example, the insertion of a transposon at many different sites in E2 still results in a viable virus (Navaratmarajah, supra).

In certain embodiments, the tag peptide may be incorporated into an E3, 6K, or E1 protein. For some purposes, the label may be incorporated into E2, but the label is not ideal for products intended for administration to human patients. Tag peptides that are short sequences (e.g., 5-30 amino acids) can be used to facilitate detection of envelope expression and their presence in the virion. For detection purposes, the tag sequence will typically be detectable by antibody or chemical. Another use of the tag is to facilitate purification of the virus particles. Substrates containing the tagged binding partner can be used to adsorb the virus. Elution of the virus can be achieved by: treatment with moieties that displace the tag from the binding partner, or when the tag sequence forms a bond with the cleavage sequence, treatment with an appropriate endopeptidase will conveniently allow release of the virus. (see, e.g., Qiagen catalog, Factor Xa Protease System). Removal of the tag peptide is often required for safety purposes of use of the virion in an animal subject. If the tag is not removed, an immune response to the tag may occur.

Suitable tags include, but are not limited to, flag (dykddddk) (U.S. Pat. No.4,703,004, incorporated in its entirety) (antibodies thereto are commercially available), chitin-binding protein, maltose-binding protein, glutathione-S-transferase, polyhistidine (U.S. Pat. No.4,569,794, incorporated in its entirety), thioredoxin, HA (hemagglutinin) -tags, among others. Polyhistidine can be adsorbed onto affinity media containing bound metal ions (e.g., nickel or cobalt) and can be eluted with low pH media.

Virions can be evaluated to determine the specificity of envelope glycoproteins incorporated into dendritic cell-targeted viruses. For example, a mixed bone marrow cell population can be obtained from a subject and cultured in vitro. Alternatively, isogenic cell lines expressing DC-SIGN or not may be obtained and used. The recombinant virus may be administered to a mixed bone marrow cell population or isogenic cell lines, and the expression of the reporter gene incorporated into the virus in the cultured cells may be determined. Certain embodiments may use limiting dilution assays in which a mixed population of cells is divided into separate fractions and then independently incubated with reduced amounts of virus (e.g., fractions with 2-fold, 5-fold, 10-fold less virus). In some embodiments, at least about 50%, more preferably at least about 60%, 70%, 80% or 90%, more preferably at least about 95% of the infected cells in the mixed cell population are DC-SIGN expressing dendritic cells. In certain embodiments, the ratio of infected dendritic cells to infected non-dendritic cells (or cells that do not express DC-SIGN) is at least about 2: 1, at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8: 1, at least about 9: 1, at least about 10: 1, at least about 20: 1, at least about 30: 1, at least about 40: 1, at least about 50: 1, at least about 100: 1, at least about 200: 1, at least about 500: 1, at least about 1000: 1, at least about 5000: 1, at least about 10,000: 1, or greater. For limiting dilution, greater selectivity is typically seen with higher dilutions (i.e., reduced amounts) of the input virus.

The activity of pseudotyped virions can be determined by any of a variety of techniques. For example, a preferred method of measuring infection efficiency (IU, infectious unit) is by administering a viral particle to a cell and measuring the expression of a product encoded in the vector genome. Any product that can be determined can be used. One convenient type of product is a fluorescent protein, such as Green Fluorescent Protein (GFP). GFP and assays are exemplified in the examples (such as example 3). Other products that may be used include proteins expressed on the cell surface (e.g., detected by antibody binding), enzymes, and the like. If the product is an antigen and the cells are dendritic cells, infectivity/activity can be assessed by measuring the immune response. In addition, side effects in mammals may be identified. The ability to specifically target dendritic cells can also be tested directly, e.g., in cell culture, as described below.

Viral particles can also be prepared and tested for their selectivity and/or their ability to facilitate penetration of a target cell membrane. Virions with an envelope with unmodified glycoproteins can be used as controls for comparison. Briefly, cells expressing receptors for envelope glycoproteins are infected by viruses using standard infection assays. After a defined period of time, e.g., 48 hours post-infection, cells can be collected and the percentage of cells infected with the virus can be determined by, e.g., flow cytometry. Selectivity can be scored by calculating the percentage of cells infected by the virus. Similarly, the effect of a variant envelope glycoprotein on viral titer can be quantified by dividing the percentage of cells infected with a virus comprising the variant envelope by the percentage of cells infected with a virus comprising the corresponding wild-type (unmodified) envelope glycoprotein. Particularly suitable variants will have the best combination of selectivity and infectious titer. Once the variants are selected, virus concentration assays can be performed to confirm that these viruses can be concentrated without compromising activity. Viral supernatants were collected and concentrated by ultracentrifugation. Viral titers can be determined by: limiting dilution of viral stock solutions and infection of cells expressing receptors for envelope glycoproteins, and measuring expression of products expressed by the viruses as described above.

Entry of lentiviral vector particles into target cells is another type of activity assessment. BlaM-Vpr (beta-lactamase Vpr) fusion proteins have been used to assess HIV-1 virus penetration; fusions of BlaM and Sindbis virus envelope glycoproteins (such as E1 or E2/E1 fusion proteins) can be used to assess the efficacy of envelope proteins in facilitating fusion and penetration into target cells. The virions can be prepared, for example, by transiently transfecting the packaging cell with one or more vectors comprising the viral element, BlaM-Vpr, and the target variant envelope (and optionally an affinity molecule). The resulting virus can be used to infect cells that express molecules to which the target molecule (or affinity molecule) will specifically bind in the absence or presence of free binding inhibitors (such as antibodies). Then, the cells can be washed with CO 2-independent medium and loaded with CCF2 dye (Aurora Bioscience). After incubation at room temperature to complete the lysis reaction, cells can be fixed by paraformaldehyde and analyzed by flow cytometry and microscopy. The presence of blue cells indicates penetration of the virus into the cytoplasm; when a blocking antibody is added, fewer blue cells will be expected (Cavrois et al Nat Biotechnol 20: 1151-1154, 2002; the entire contents of which are incorporated).

To investigate whether penetration depends on low pH and identify envelope glycoproteins with the desired pH dependence, NH4Cl or other compounds that alter pH (NH4Cl will neutralize the acidic compartment of the endosome) may be added at the infection step. In the case of NH4Cl, the disappearance of blue cells would indicate that viral penetration is low pH-dependent. In addition, to confirm that the activity is pH-dependent, lysotropic agents such as ammonium chloride, chloroquine, concanavalin (concanamycin), bafilomycin aluminum (bafilomycin Al), monensin (monensin), nigericin (nigericin), and the like may be added to the incubation buffer. These agents increase the pH of the lumen of the endosome (e.g., Drose and Altendorf, j.exp. biol.200, 1-8, 1997). Inhibition of these agents will reveal the effect of pH on viral fusion and entry. Different entry kinetics between viruses displaying different fusion gene molecules can be compared and the most suitable virus selected for a particular application.

PCR-based entry assays can be used to monitor reverse transcription and measure the kinetics of viral DNA synthesis as an indication of viral entry kinetics. For example, virions containing a particular envelope protein molecule are incubated with target cells, such as 293T cells, DCs, or any other cells that have been engineered to express or naturally express an appropriate binding partner (receptor) for the envelope protein molecule. Immediately or after an amount of time (to allow infection to occur) unbound virus is removed and an aliquot of the cells is analyzed for viral nucleic acid. DNA was extracted from these aliquots and subjected to amplification analysis, which was typically primed with LTR-specific primers in a semi-quantitative assay. The appearance of LTR-specific DNA products indicates the success of viral entry.

B. Lentiviral vector genomes

The viral vector particle comprises a genome comprising a sequence of interest. Other sequences may include, for example, sequences that allow for packaging of the genome into a virion and sequences that facilitate expression of the sequence of interest following transduction of a target cell. The genome may be derived from any of a number of suitable, available lentiviral genome-based vectors, including those identified for human gene therapy applications, such as those described by Pfeifer and Verma (Annu. Rev. genomics hum. Genet. 2: 177-211, 2001; the entire contents of which are incorporated herein by reference). For simplicity, the genome is also referred to as a "viral vector genome" or a "vector genome".

1. Framework

Suitable lentiviral vector genomes include those based on the following viruses: human immunodeficiency virus (HIV-1), HIV-2, Feline Immunodeficiency Virus (FIV), equine infectious anemia virus, Simian Immunodeficiency Virus (SIV), and Meddi/visna virus (maedi/visna virus). Desirable characteristics of lentiviruses are that they are capable of infecting both dividing and non-dividing cells, and that the target cells need not be dividing (or have to be stimulated to divide). Typically, the genomic and envelope glycoproteins will be based on different viruses so that the resulting viral vector particle is pseudotyped. The safety features of the vector genome are ideally incorporated. Safety features include self-inactivating LTRs and non-integrating genomes. Exemplary vectors are further discussed in example 5 and fig. 5, and such vectors may be used in embodiments of the invention to express an antigen of interest.

In certain exemplary embodiments, the viral vector genome comprises sequences from a lentiviral genome (such as an HIV-1 genome or an SIV genome). The viral genome construct may comprise sequences from the 5 'and 3' LTRs of a lentivirus, and in particular, may comprise R and U5 sequences from the 5 'LTR of a lentivirus and an inactivated or self-inactivating 3' LTR from a lentivirus. The LTR sequence may be an LTR sequence from any lentivirus of any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequence is an HIV LTR sequence.

The vector genome may comprise an inactivated or self-inactivated 3' LTR (Zufferey et al J Virol 72: 9873, 1998; Miyoshi et al J Virol 72: 8150, 1998; both incorporated in their entirety). Self-inactivating vectors typically have deletions of enhancer and promoter sequences from the 3 'Long Terminal Repeat (LTR) that is replicated into the 5' LTR during vector integration. In one case, the U3 element of the 3' LTR contains deletions of its enhancer sequence, TATA box, Sp1, and NF-. kappa.B sites. As a result of self-inactivation of the 3'LTR, the provirus produced after entry and reverse transcription will contain an inactivated 5' LTR. The rationale is to improve safety by reducing the risk of activity of the vector genome and the effect of LTRs on nearby cellular promoters. The self-inactivating 3' LTR may be constructed by any method known in the art.

Optionally, the U3 sequence from the lentiviral 5' LTR may be replaced with a promoter sequence in the viral construct (such as a heterologous promoter sequence). This increases the titer of virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line can be used. In one embodiment, a CMV enhancer/promoter sequence is used (US5385839 and US 5168062, the entire contents of each of which are incorporated).

In certain embodiments, the risk of insertional mutagenesis occurring is minimized by constructing the integration-deficient lentiviral vector genome. Various methods can be pursued to generate non-integrative vector genomes. These methods require engineering mutations into the integrase component of the pol gene so that it encodes a protein with inactivated integrase. The vector genome itself may be modified to prevent integration by, for example, mutating or deleting one or both of the junction sites, or rendering the 3' LTR proximal polypurine channel (PPT) non-functional via deletion or modification. In addition, non-genetic methods are available; these methods include agents that inhibit one or more functions of integrase. The methods described are not mutually exclusive, that is, more than one of the methods may be used at a time. For example, both the integrase and the ligation site may be non-functional, or the integrase and the PPT site may be non-functional, or the ligation site and the PPT site may be non-functional or all of them may be non-functional.

As mentioned above, one approach is to prepare and use a non-functional integrase. Integrase is involved in the cleavage of the viral double-stranded blunt-ended DNA and in the ligation of the ends to the 5' phosphates in both strands of the chromosomal target site. Integrases have three functional domains: an N-terminal domain comprising a zinc binding motif (HHCC); a central domain nucleus containing the catalytic nucleus and the conserved DD35E motif (D64, D116, E152 in HIV-1); and a C-terminal domain having DNA binding properties. The point mutations introduced into integrase are sufficient to disrupt normal function. A number of integrase mutations have been constructed and characterized (see, Philpott and Thrasher, Human Gene Therapy 18: 483, 2007; Apolonia, Thesis submitted to University College London, 2009, 4, pages 82-97; Engelman et al J Virus 69: 2729, 1995; Nightingale et al Mol Therapy, 13: 1121, 2006; all of these references are incorporated in their entirety). The sequence encoding the integrase protein may be deleted or mutated to inactivate the protein, preferably without impairing reverse transcriptase activity or nuclear targeting, and thus only preventing provirus integration into the target cell genome. Acceptable mutations may reduce integrase catalytic function, strand transfer function, binding to att sites, binding to host chromosomal DNA, and other functions. For example, a single aspartate to asparagine substitution at residue 35 of HIV or SIV integrase completely abolishes viral DNA integration. Deletion of integrase will generally be limited to the C-terminal domain. Deletion of the coding sequence for residue 235-288 results in a useful non-functional integrase (Engelman et al J Virol 69: 2729, 1995). As additional examples, mutations can be made, such as Asp64 (residue numbering for HIV-1, corresponding residue numbering for integrases from other lentiviruses or retroviruses can be readily determined by the ordinarily skilled artisan) (e.g., D64E, D64V), Asp116 (e.g., D116N), Asn120 (e.g., N120K), Glu152, gin 148 (e.g., Q148A), Lys156, Lys159, Trp235 (e.g., W235E), Lys264 (e.g., K264R), Lys266 (e.g., K266R), Lys273 (e.g., K273R). Other mutations can be constructed and tested for integration, transgene expression, and any other desired parameters. The determination of these functions is well known. Mutations can be generated by any of a variety of techniques, including site-directed mutagenesis of nucleic acid sequences and chemical synthesis. One mutation may be made, or more than one of these mutations may be present in the integrase. For example, an integrase may have mutations at two amino acids, three amino acids, four amino acids, and the like.

Alternatively or in combination with the use of integrase mutants, the linkage sites (att) in U3 and U5 may also be mutated. Integrases bind to these sites and the 3' terminal dinucleotide cleaves at both ends of the vector genome. The CA dinucleotide is located at the 3' concave end; CA is essential for processing, and mutations in nucleotides block integration into the host chromosome. A of the CA dinucleotide is the most critical nucleotide for integration, and mutations at both ends of the genome will give the best results (Brown et al J Virol 73: 9011 (1999)). In one example, the CA at each end is changed to TG. In other examples, the CA at each end is changed to TG at one end and GT at the other end. In other examples, the CA at each end is deleted; in other examples, the a of the CA at each end is deleted.

Integration can also be inhibited by mutation or deletion of the polypurine channel (PPT) located proximal to the 3' LTR (WO 2009/076524; incorporated in its entirety). PPT is a polypurine sequence of about 15 nucleotides that can be used as a primer binding site for plus strand DNA synthesis. In this case, mutations or deletions of PPT target the reverse transcription process. Without wishing to be limited to a certain mechanism of mutation or deletion of PPT, the production of linear DNA is mainly reduced and essentially only the 1-LTR DNA loop is produced. Integration requires a linear double-stranded DNA vector genome and substantially eliminates integration of the genome without a linear double-stranded DNA vector. As mentioned above, PPT may be made non-functional by mutation or by deletion. Typically, the entire about 15nt of PPT is deleted, but in some embodiments, shorter deletions of 14nt, 13nt, 12nt, 11nt, 10nt, 9nt, 8nt, 7nt, 6nt, 5nt, 4nt, 3nt, and 2nt may be made. When mutations are made, multiple mutations are usually made, particularly in the 5' half of PPT, although single and double mutations in the first four bases still reduce transcription (McWilliams et al, JVirol 77: 11150, 2003). Mutations made at the 3' end of PPT usually have a more pronounced effect (Powell and Levin J Virol 70: 5288, 1996).

These different methods of rendering the vector genome non-integrated may be used alone or in combination. More than one method may be used to construct a reliable carrier (fail-safe vector) via a redundancy mechanism. Thus, a PPT mutation or deletion may be combined with an att site mutation or deletion or with an integrase mutation, or a PPT mutation or deletion may be combined with both an att site mutation or deletion and an integrase mutation. Similarly, att site mutations or deletions and integrase mutations may be combined with each other in combination with PPT mutations or deletions.

2. Regulatory element

As discussed herein, the viral vector genome comprises a sequence of interest that is desired to be expressed in a target cell. Typically, the target sequence is located between the 5 'LTR and 3' LTR sequences. In addition, the target sequence is preferably functionally associated with other genetic elements (e.g., transcriptional regulatory sequences including promoters or enhancers) to regulate expression of the target sequence in a particular manner. In some cases, useful transcriptional regulatory sequences are those that are highly regulated in time and space with respect to activity. Expression control elements useful for regulating expression of a component are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signal sequences, enhancers and other regulatory elements.

The sequence of interest and any other expressible sequence are usually functionally related to internal promoter/enhancer regulatory sequences. An "internal" promoter/enhancer is a promoter/enhancer positioned between the 5 'LTR sequence and the 3' LTR sequence of a viral vector construct and operably linked to a sequence of interest. An internal promoter/enhancer can be any promoter, enhancer, or promoter/enhancer combination known to increase expression of a gene with which it is functionally associated. "functional relationship" and "operably linked" means, but is not limited to, that the sequences are in the correct position and orientation relative to the promoter and/or enhancer such that the sequence of interest is expressed when the promoter and/or enhancer is contacted with the appropriate molecule.

The choice of internal promoter/enhancer is based on the desired expression pattern of the target sequence and the specific properties of known promoters/enhancers. Thus, an internal promoter may be constitutively active. Non-limiting examples of constitutive promoters that can be used include promoters from: ubiquitin (US 5510474; WO 98/32869, the entire contents of each of which are incorporated herein by reference), CMV (Thomsen et al, PNAS 81: 659, 1984; US 5168062, the entire contents of each of which are incorporated herein by reference), β -actin (Guining et al, 1989 Proc. Natl. Acad. Sci. USA 84: 4831-4835, the entire contents of which are incorporated herein by reference), and pgk (see, e.g., Adra et al, 1987 Gene 60: 65-74; Singer-Sam et al, 1984 Gene 32: 409-417; and Dobson et al, 1982 Nucleic Acids Res.10: 2635-2637, the entire contents of each of which are incorporated herein by reference).

Alternatively, the promoter may be a tissue-specific promoter. In some preferred embodiments, the promoter is a target cell-specific promoter. For example, the promoter may be from any product expressed by dendritic cells, including CD11c, CD103, TLR, DC-SIGN, BDCA-3, DEC-205, DCIR2, mannose receptor, Dectin-1, Clec9A, MHC class II. In addition, the promoter may be selected to allow inducible expression of the sequence of interest. Many systems for inducible expression are known in the art, including the tetracycline response system, the lac operator-suppressor subsystem, and promoters responsive to a variety of environmental or physiological changes, including heat shock, metal ions (such as the metallothionein promoter), interferons, hypoxia, steroids (such as the progesterone or glucocorticoid receptor promoter), radiation (such as the VEGF promoter). Combinations of promoters may also be used to obtain the desired expression of the gene of interest. One of ordinary skill in the art will be able to select a promoter based on the desired gene expression pattern in the target organism or cell.

The viral genome may comprise at least one RNA polymerase II or III reactive promoter. Such promoters may be operably linked to the target sequence and may also be linked to termination sequences. In addition, more than one RNA polymerase II or III promoter may be incorporated. RNA polymerase II and III promoter is the technicians in this field is known. Suitable ranges for RNA polymerase III promoters can be found, for example, in Paule and White, Nucleic Acids research, Vol.28, pp.1283-1298 (2000), the entire contents of which are incorporated herein by reference. RNA polymerase II or III promoter also includes any synthetic or engineered DNA fragment that can direct transcription of a downstream RNA coding sequence by RNA polymerase II or III. In addition, the RNA polymerase II or III (Pol II or III) promoter used as part of the viral vector genome may be inducible. Any suitable inducible Pol II or III promoter may be used with the methods of the invention. Particularly suitable Pol II or III promoters include the tetracycline-responsive promoters provided in Ohkawa and Taira, Human Gene Therapy, Vol.11, pp.577-585 (2000) and Meissner et al Nucleic acids research, Vol.29, pp.1672-1682 (2001), the entire contents of each of which are incorporated herein by reference.

Internal enhancers may also be present in the viral construct to enhance expression of the gene of interest. For example, the CMV enhancer may be used (Boshart et al Cell, 41: 521, 1985; the entire contents of which are incorporated herein by reference). Many enhancers in viral genomes (such as HIV, CMV) and mammalian genomes have been identified and characterized (see gene bank). Enhancers may be used in combination with heterologous promoters. One of ordinary skill in the art will be able to select an appropriate enhancer based on the desired expression pattern.

The viral vector genome will typically contain a promoter that is recognized by the target cell and operably linked to the sequences of interest, viral components, and other sequences discussed herein. A promoter is an expression control element formed from a nucleic acid sequence that allows binding and transcription of RNA polymerase to occur. Promoters may be inducible, constitutive, temporally active, or tissue specific. The activity of an inducible promoter is induced by the presence or absence of biological or non-biological agents. Inducible promoters can be a useful tool in genetic engineering because the expression of the genes to which they are operably linked can be initiated or halted at a particular developmental stage of an organism, at the time of its manufacture, or in a particular tissue. Inducible promoters can be divided into chemically regulated promoters and physically regulated promoters. Typical chemically regulated promoters include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase i (alca) gene promoters), tetracycline regulated promoters (e.g., tetracycline responsive promoters), steroid regulated promoters (e.g., rat Glucocorticoid Receptor (GR) -based promoters, human Estrogen Receptor (ER) -based promoters, moth ecdysone receptor-based promoters, and steroid/retinol/thyroid receptor superfamily-based promoters), metal regulated promoters (e.g., metallothionein gene-based promoters), and pathogen-associated promoters (e.g., Arabidopsis thaliana (Arabidopsis) and maize pathogen-associated (PR) -based proteins). Typical physically regulated promoters include, but are not limited to, temperature regulated promoters (e.g., heat shock promoters) and light regulated promoters (e.g., soybean SSU promoters). Other exemplary Promoters are described elsewhere, for example, "Promoters used to regulate expression" on Patent Lens web site accessed on day 18, 5/2009 (which is incorporated by reference herein in its entirety).

One skilled in the art will be able to select the appropriate promoter based on the particular circumstances. Many different promoters are well known in the art, as well as methods for operably linking the promoter to the gene to be expressed. Both native promoter sequences and many heterologous promoters can be used to direct expression in packaging cells and target cells. However, heterologous promoters are preferred because they generally allow for greater transcription and higher yields of the desired protein compared to the native promoter.

Promoters may, for example, be obtained from the genome of viruses such as polyoma virus, avian bean virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Simian Virus 40(SV 40). The promoter may also be, for example, a heterologous mammalian promoter, such as an actin promoter or an immunoglobulin promoter, a heat shock promoter, or a promoter normally associated with a native sequence, provided that such a promoter is compatible with the target cell. In one embodiment, the promoter is a naturally occurring viral promoter in a viral expression system. In certain embodiments, the promoter is a dendritic cell-specific promoter. The dendritic cell-specific promoter can be, for example, the CD11c promoter.

Transcription can be enhanced by inserting enhancer sequences into the vector. Enhancers are generally cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin) and from eukaryotic viruses. Examples include the SV40 enhancer on the posterior side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the origin of replication, and adenovirus enhancers. The enhancer may be spliced into the vector at the 5 ' or 3' position of the antigen specific polynucleotide sequence, but is preferably located at the 5 ' position of the promoter.

The expression vector may also contain sequences necessary for termination of transcription and for stabilization of the mRNA. These sequences are often found in the 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA and are well known in the art.

The viral vector genome may also contain additional genetic elements. The type of elements that can be included in the constructs is not limited in any way and can be selected to achieve a particular result. For example, a signal sequence that facilitates nuclear entry of the viral genome in the target cell can be included. An example of such a signal sequence is the HIV-1flap signal sequence. In addition, elements may be included that facilitate the characterization of proviral integration sites in the target cell. For example, a tRNA amber suppressor gene sequence can be included in the construct. Insulator sequences from, for example, chicken beta-globin can also be included in the viral genome construct. This element reduces the chance of silencing of the integrative provirus in the target cell due to methylation and heterochromatin. In addition, insulators can protect internal enhancers, promoters and exogenous genes from the positive or negative positional effects of surrounding DNA at the integration site on the chromosome. In addition, the vector genome may contain one or more genetic elements designed to enhance expression of the gene of interest. For example, woodchuck hepatitis virus-responsive elements (WREs) can be placed in constructs (Zufferey et al 1999. J.Virol.74: 3668-.

Viral vector genomes are usually constructed in the form of plasmids that can be transfected into packaging or producer cell lines. Plasmids typically contain sequences for replicating the plasmid in bacteria. Such plasmids are well known in the art. In addition, vectors comprising a prokaryotic origin of replication may also comprise a gene expressing a detectable or selectable marker, such as drug resistance. Typical bacterial drug resistance products are those which confer ampicillin or tetracycline resistance.

Plasmids containing one or more of the components described herein are readily constructed using standard techniques well known in the art. For analysis to confirm the correct sequence in the constructed plasmid, the plasmid can be replicated in E.coli (E.coli), purified and analyzed by restriction endonuclease digestion or its DNA sequence determined by conventional methods.

Vectors constructed for transient expression in mammalian cells may also be used. Transient expression involves the use of expression vectors that are capable of efficient replication in a host cell, such that the host cell accumulates many copies of the expression vector and then synthesizes high levels of the polypeptide encoded by the antigen-specific polynucleotide in the expression vector. See, Sambrook et al, supra, pages 16.17-16.22. Other vectors and methods suitable for adapting to the expression of polypeptides are well known in the art and are readily adapted to the particular circumstances.

Using the teachings provided herein, one skilled in the art will recognize that the efficacy of a particular expression system can be tested by transfecting a packaging cell with a vector comprising a gene encoding a reporter protein and measuring expression using an appropriate technique (e.g., measuring fluorescence in a green fluorescent protein conjugate). Suitable reporter genes are well known in the art.

3. Type of target sequence

The sequence of interest is not limited in any way and includes any nucleic acid that the ordinarily skilled artisan desires to integrate, transcribe and express in the target cell. The product may be a protein or a nucleic acid. The target sequence may encode a protein or nucleic acid molecule comprising an siRNA, a microrna, a self-complementary double-stranded RNA having a complementary region length of greater than about 20 ribonucleotides, or an RNA complementary to a messenger RNA, wherein binding of the complementary (antisense) RNA to the messenger RNA blocks its ability to be translated into a protein. In some cases, the sequence of interest may encode an antigen against which an immune response is desired. In particular, tumor antigens and infectious disease antigens from diseases such as HIV, HSV, HCV, HPV, malaria or tuberculosis are desirable.

In certain instances, the target sequence may be a gene encoding a target small inhibitory rna (sirna) or microrna (mirna) that down-regulates the expression of the molecule. For example, genes encoding siRNA or microrna can be used to down-regulate expression of negative regulators in cells, including those that inhibit activation or maturation of dendritic cells. siRNAs and microRNAs are well known in The art (Fire et al, Nature 391: 806, 1998; see also "The RNA Interference Resource" of applied Biosystems, Trang et al, Oncogene Suppl 2: S52, 2008; Taganov, K. et al 2007.Immunity 26: 133-137; Dahlberg, J.E. and E.Lund.2007.Sci.STKE 387: pe 25; Tiemann and Rossi, EMBO Mol Med 1: 142, 2009). Alternatively, the target sequence may encode a self-complementary double-stranded RNA having a complementarity region greater than about 20 ribonucleotides in length or an antisense RNA having a length greater than about 20 ribonucleotides in length. One of ordinary skill in the art will appreciate that siRNA, miRNA, dsRNA and antisense RNA molecules can be expressed from an RNA polymerase III promoter or can be a component of a non-coding RNA transcribed from an RNA polymerase II promoter.

In addition, the target sequence may encode more than one product. In some configurations, the sequence to be delivered may comprise a plurality of genes encoding at least one protein, at least one siRNA, at least one microrna, at least one dsRNA, or at least one antisense RNA molecule, or any combination thereof. For example, the sequence to be delivered may include one or more genes encoding one or more antigens against which an immune response is desired. One or more antigens may be associated with a single disease or condition, or they may be associated with multiple diseases and/or conditions. In some cases, the gene encoding the immune modulatory protein can be included along with the gene encoding the antigen against which an immune response is desired, and such a combination can elicit and modulate an immune response in the desired direction and magnitude. In other cases, sequences encoding siRNA, microrna, dsRNA, or antisense RNA molecules can be constructed with genes encoding antigens against which an immune response is desired, and such combinations can modulate the scope of the immune response. The product may be produced as an initial fusion product in which the coding sequence is functionally associated with a promoter. Alternatively, the products may be encoded independently and each coding sequence functionally related to the promoter. The promoters may be the same or different.

In certain configurations, the vector comprises a polynucleotide sequence encoding a dendritic cell maturation/stimulation factor. Exemplary stimulatory molecules include GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand (CD40L), drug-inducible CD40(iCD40), and the like. These polynucleotides are typically under the control of one or more regulatory elements that direct the expression of the coding sequence in the dendritic cell. Maturation of dendritic cells facilitates successful inoculation (Banchereau, J and Palucka, A.K. Nat. Rev. Immunol.5: 296-306 (2005); Schuler, G. et al, curr. Opin. Immunol.15: 138-147 (2003); Figdor, C.G. et al, nat. Med.10: 475-480 (2004)). Maturation can transform DCs from cells actively involved in capturing antigens in cells specialized for T cell priming. For example, binding of CD40 to CD 4-helper T cells via CD40L is a critical signal for DC maturation, resulting in efficient activation of CD8T cells. Such stimulatory molecules are also known as maturation factors or maturation stimulatory factors. Immune checkpoints represent an important barrier to activation of functional cellular immunity in cancer, and antagonistic antibodies specific for inhibitory ligands on T cells (including CTLA4 and programmed death-1 (PD-1) are examples of clinically evaluated targeting agents.an important tolerance mechanism in chronic infection and cancer is functional depletion of Ag-specific T cells expressing high levels of PD-1. the efficacy of therapeutic immunity has been shown to be significantly enhanced by combination with immune checkpoint control (as a non-limiting example), it will be appreciated by those of ordinary skill in the art that an alternative method of inhibiting immune checkpoints is to inhibit expression of Programmed Death (PD) ligands 1 and 2 (PD-L1/L2). one way to achieve inhibition is expression of RNA molecules such as those described herein, which inhibits expression of PD-L1/L2 in DCs transduced by lentiviral vectors encoding one or more RNA molecules Maturation or expression of specific elements such as immune checkpoints (e.g., PD-1 ligands) can be characterized by flow cytometry analysis of upregulation of surface markers such as MHC II and profiles of expressed chemokines and cytokines.

Sequences encoding detectable products (typically proteins) can be included to allow identification of cells expressing the desired product. For example, a fluorescent marker protein such as Green Fluorescent Protein (GFP) is incorporated into the construct along with the sequence of interest (e.g., the sequence encoding the antigen). In other cases, the protein may be detected by an antibody, or the protein may be an enzyme that acts as a substrate, so as to produce a detectable product or a product that allows selection for transfection or transduction of a target cell (e.g., conferring drug resistance, such as hygromycin resistance). The proteins encoded by typical selection genes confer resistance to antibiotics or other toxins suitable for use in eukaryotic cells, such as neomycin, methotrexate, blasticidin (blesidine), among others known in the art, or supplement auxotrophic deficiencies, or supply critical nutrients retained from the culture medium. The selectable marker may optionally be present on a separate plasmid and introduced by co-transfection.

One or more polycistronic expression units comprising two or more of the elements (e.g., target sequences, envelope molecules, DC maturation factors) necessary for production of the desired virus in the packaging cell can be used. The use of polycistronic vectors reduces the total number of nucleic acid molecules required and thus avoids the potential difficulties associated with the coordinated expression from multiple vector genomes. In a polycistronic vector, the various elements to be expressed are operably linked to one or more promoters (and other expression control elements as desired). In certain configurations, the polycistronic vector comprises a sequence of interest, a sequence encoding a reporter product, and a viral element. The sequence of interest typically encodes an antigen and optionally a DC maturation factor. Sometimes, the polycistronic vector contains a gene encoding an antigen, a gene encoding a DC maturation factor, and viral elements.

The components to be expressed in the polycistronic expression vector may be separated, for example, by an Internal Ribosome Entry Site (IRES) element or a viral 2A element, to allow independent expression of the various proteins from the same promoter. IRES elements and 2A elements are known in the art (U.S. Pat. No.4,937,190; de Felipe et al 2004.Traffic 5: 616-626, each of which is incorporated herein by reference in its entirety). In one embodiment, oligonucleotides encoding furin cleavage site sequences (RAKR) (Fang et al 2005.nat. Biotech 23: 584-590, the entire contents of which are incorporated herein by reference) linked to 2A-like sequences from Foot and Mouth Disease Virus (FMDV), Equine Rhinitis A Virus (ERAV) and Spodoptera litura virus (TaV) (Szymczak et al 2004.nat. Biotechnol.22: 589594, the entire contents of which are incorporated herein by reference) are used to separate the genetic elements in a polycistronic vector. The efficacy of a particular polycistronic vector can be readily tested by detecting the expression of each gene using standard protocols.

In a particular example, the viral vector genome comprises: cytomegalovirus (CMV) enhancer/promoter sequences; r and U5 sequences from the HIV 5' LTR; a packaging sequence (ψ); an HIV-1flap signal sequence; an internal enhancer; an internal promoter; a target gene; woodchuck hepatitis virus reactive element; a tRNA amber suppressor gene sequence; deletion of the U3 element of the enhancer sequence; chicken beta-globulin insulators; and the R and U5 sequences of the 3' HIV LTR. In some examples, the vector genome comprises the complete lentiviral 5 'LTR and the self-inactivating 3' LTR. (Iwakuma et al Virology 15: 120, 1999, which is incorporated by reference in its entirety).

Construction of the vector genome can be accomplished using any suitable genetic engineering technique known in the art, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, such as, for example, Sambrook et al (1989.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y.), coffee et al (retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997)) and "RNA Viruses: standard techniques described in a Practical Approach "(Alan j. cann, ed., oxford university Press, (2000)), the entire contents of each of the foregoing references being incorporated herein by reference.

4. Preparation of viral particles

Any of a variety of methods known in the art can be used to prepare infectious lentiviral particles whose genomes comprise an RNA copy of the viral vector genome. In one method, the viral vector genome is introduced into a packaging cell line containing all the components necessary for packaging the viral genomic RNA, which is transcribed from the viral vector genome into viral particles. Alternatively, the viral vector genome may comprise one or more genes encoding viral components in addition to one or more sequences of interest. However, to prevent genome replication in the target cell, the endogenous viral genes required for replication are typically removed and provided separately in the packaging cell line.

Generally, lentiviral vector particles are prepared by transfecting a cell line with one or more plasmid vectors containing the components necessary to produce the particles. These lentiviral vector particles are generally not replication competent, that is, they are only capable of a single round of infection. Most often, multiple plasmid vectors are used to separate the various genetic components that produce lentiviral vector particles, which primarily reduces the chance of recombination events that might otherwise produce replication-competent viruses. However, a single plasmid vector with all lentiviral components can be used, if desired. As an example of a system using multiple plasmid vectors, cell lines were transfected with: at least one plasmid containing a viral vector genome (i.e., a vector genome plasmid) comprising LTRs, cis-acting packaging sequences, and a target sequence, often operably linked to a heterologous promoter; at least one plasmid encoding a viral enzymatic and structural component (i.e., a packaging plasmid encoding a component such as Gag and Pol); and at least one envelope plasmid encoding an envelope glycoprotein of an arbovirus. Additional plasmids (e.g., Rev expression plasmids) as described herein and known in the art can be used to enhance retroviral particle production. The virion buds through the cell membrane and comprises a nucleus containing a genome containing the sequence of interest and an arbovirus envelope glycoprotein that targets dendritic cells. When the arbovirus glycoprotein is sindbis virus E2 glycoprotein, the glycoprotein is engineered to reduce binding to heparan sulfate compared to the reference strain HR.

Transfection of packaging cells with the plasmid vectors of the present invention can be accomplished by well-known methods, and the method used is not limited in any way. Many Non-viral delivery systems are known in the art, including, for example, electroporation, lipid-based delivery systems including liposomes, "naked" DNA delivery, and delivery of polycyclodextrin compounds using compounds such as those described in Schatzlein AG (2001.Non-viral vectors in Cancer Gene Therapy: Principles and procedures. Cationic lipid or salt treatment methods are commonly used, see, e.g., Graham et al (1973. Virol.52: 456); wigler et al (1979.Proc. Natl. Acad. Sci. USA 76: 1373-76), the entire contents of each of the foregoing references being incorporated herein by reference. Calcium phosphate precipitation is most commonly used. However, other methods of introducing the vector into the cell may also be used, including nuclear microinjection and bacterial protoplast fusion.

Packaging cell lines provide components including viral regulatory and structural proteins that need to be reversed for packaging of viral genomic RNA into lentiviral vector particles. The packaging cell line can be any cell line capable of expressing lentiviral proteins and producing functional lentiviral vector particles. Some suitable packaging cell lines include 293(ATCC CCLX), 293T, HeLa (ATCC CCL 2), D17(ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cells. The packaging cell line stably expresses the necessary viral proteins. Such packaging cell lines are described, for example, in U.S. patent No. 6,218,181, which is incorporated herein by reference in its entirety. Alternatively, a packaging cell line can be transiently transfected with a nucleic acid molecule encoding one or more essential viral proteins along with a viral vector genome. The resulting virus particles are collected and used to infect target cells. The genes encoding the envelope glycoproteins are typically cloned into expression vectors such as pcDNA3(Invitrogen, CAUSA). Eukaryotic expression vectors are well known in the art and are available from many commercial sources. Packaging cells, such as 293T cells, are then co-transfected with a viral vector genome encoding a sequence of interest, typically a sequence encoding an antigen, at least one plasmid encoding a viral packaging component, and a vector for expressing the target molecule. The envelope is expressed on the membrane of the packaging cell and incorporated into a viral vector.

In one instance, one or more vectors are used to introduce polynucleotide sequences into a packaging cell line in order to prepare lentiviral vector particles pseudotyped with sindbis virus envelope glycoproteins (such as E2) as described herein. The vector may contain polynucleotide sequences encoding various components of the virus, including the sindbis virus envelope, the sequence of interest (typically the sequence encoding the antigen), and any components necessary for the production of the virus that are not provided by the packaging cell.

In other cases, the packaging cell is co-transfected with the viral vector genome encoding the antigen and one or more additional vectors. For example, in addition to the viral vector encoding the antigen, the second vector preferably carries a gene encoding a modified (also referred to as variant) sindbis viral envelope. In some cases, the viral vector genome encoding the antigen also includes a polynucleotide sequence encoding a selected immune modulator, including by way of non-limiting example, a chemokine, a cytokine, a DC maturation factor, or a factor that modulates an immune checkpoint mechanism. In other cases, the polynucleotide sequence encoding the selected immune modulator is contained in a third vector, which is co-transfected into a packaging cell with the viral vector encoding the antigen and one or more additional vectors.

Production of the Virus is measured as described herein and expressed as IU per volume IU.IU is an infectious unit, or Transduction Unit (TU). IU and TU can be used interchangeably as a quantitative measure of potency of preparations of viral vector particles As described herein, the genome in the prepared viruses can express an easily measurable product⁵IU/mL cell supernatant of at least 3 × 10⁵IU/mL cell supernatant of at least 1 × 10⁶IU/mL cell supernatant of at least 3 × 10⁶IU/mL cell supernatant or at least 1 × 10⁷IU/mL cell supernatant (before any concentration). Alternatively, the titer is at least 80%, at least 90%, at least 95%, at least 100% of the titer of the same lentiviral vector pseudotyped with a VSV-G envelope in the same cell.

C. Delivery of viruses

The virus can be delivered to the target cell in any manner that allows the virus to contact the target Dendritic Cell (DC) where delivery of the polynucleotide of interest is desired. Sometimes a suitable amount of virus is introduced directly (in vivo) into a human or other animal, for example, via injection into the body. Suitable animals include, but are not limited to, horses, dogs, cats, cows, pigs, sheep, rabbits, chickens, or other birds. The viral particles can be injected by a number of routes, such as intravenous, intradermal, subcutaneous, intranodal, intraperitoneal, or mucosal. Viruses can be delivered using a true hypodermic device, such devices being disclosed in U.S. patent nos. 7,241,275, 7,115,108, 7,108,679, 7,083,599, 7,083,592, 7,047,070, 6,971,999, 6,808,506, 6,780,171, 6,776,776, 6,689,118, 6,670,349, 6,569,143, 6,494,865, 5,997,501, 5,848,991, 5,328,483, 5,279,552, 4,886,499, the entire contents of all of which are incorporated by reference. Other injection sites are also suitable, such as direct injection into an organ containing the target cells. For example, an intra-lymph node injection, an intra-spleen injection, or an intra-medullary injection may be used to deliver the virus to the lymph nodes, spleen, and bone marrow, respectively. Depending on the particular environment and the nature of the target cell, introduction may be via other means, including, for example, inhalation or direct contact with epithelial tissue (e.g., those in the eye, mouth, or skin).

Alternatively, the target cell is provided and contacted with the virion ex vivo, such as in a culture plate. The target cell is typically a cell population comprising dendritic cells obtained from a healthy subject or a subject in need of treatment or in need of stimulation of an immune response to an antigen. Methods of obtaining cells from a subject are well known in the art and include phlebotomy, surgical resection, and biopsy. Human DCs can also be generated by obtaining CD34a + artificial blood progenitor cells and using in vitro culture methods, as described elsewhere (e.g., Banchereau et al Cell 106, 271-274(2001) incorporated by reference in its entirety).

The virus may be suspended in culture medium and added to wells, tubes, or other containers of a culture plate. The virus-supplemented medium may be contained prior to inoculation of the cells or after the cells have been inoculated. The cells are typically incubated in an appropriate amount of medium to provide viability, and the appropriate concentration of virus in the medium is allowed to allow transduction of the host cells to occur. Preferably, the cells are incubated with the virus for a sufficient amount of time to allow the virus to infect the cells. Preferably, the cells are incubated with the virus for at least 1 hour, at least 5 hours, or at least 10 hours.

When the target cells are to be cultured, the concentration of virions is typically at least 1 IU/. mu.L, more preferably at least 10 IU/. mu.L, even more preferably at least 300 IU/. mu.L, even more preferably at least 1 × 10 IU/. mu.L⁴IU/. mu.L, even more preferably at least 1 × 10⁵IU/. mu.L, even more preferably at least 1 × 10⁶IU/. mu.L or even more preferably at least 1 × 10⁷IU/μL。

Following infection with the virus in vitro, the target cells can be introduced (or reintroduced) into a human or other animal. The cells may be introduced into the dermis, into the dermis or into the surrounding blood stream. The cells introduced into the animal are preferably of animal origin to avoid adverse immune reactions. Cells derived from donors with similar immunological backgrounds may also be used. Other cells that may also be used include those designed to avoid adverse immune responses.

For example, the target cell can be analyzed for integration, transcription, and/or expression of the sequence or gene of interest, the number of copies of the integrated gene, and the location of integration. Such analysis may be performed at any time and by any method known in the art.

The location of the infected cells of the subject to which the virus or virus-infected dendritic cells are administered, the expression of the polynucleotide or gene delivered by the target virus, the stimulation of the immune response can be analyzed and the symptoms associated with the disease or disorder monitored by any method known in the art.

The above disclosed methods of infecting cells do not depend on the individual specific characteristics of the cells. Thus, these methods are readily applicable to a variety of animal species. In some cases, the virions are delivered to human or human dendritic cells, and in other cases they are delivered to an animal, such as a mouse, horse, dog, cat, or mouse, or to avians. As discussed herein, viral vector genomes are pseudotyped to confer a broad host range as well as target cell specificity thereto. Those skilled in the art will also recognize the appropriate internal promoters and other elements to achieve the desired expression of the sequence of interest in a particular animal species. Thus, one skilled in the art would be able to modify the method of infecting dendritic cells from any species.

D. Therapeutic and prophylactic immunization

Dendritic cells can be infected with lentiviral vector particles as described herein to prevent or treat diseases or disorders, particularly those in which activation of the immune response in a patient is beneficial. Many such diseases are well known. For example, diseases or conditions that can be treated or prevented by the methods of the invention include, but are not limited to, cancer, autoimmune diseases and infections, including viral infections, bacterial infections, fungal infections, and parasitic infections. In one method, a disease is treated by the viral particles described herein to deliver a target sequence to a dendritic cell, wherein expression of the target sequence produces a disease-specific antigen and causes stimulation of antigen-specific cellular and humoral immune responses. Typically, the sequence of interest encodes an antigen against which an immune response is desired, but is not typically expressed in dendritic cells. Antigens are expressed and presented by dendritic cells. The viral vector genome may further encode a DC maturation factor.

In typical usage, the virion delivers to a dendritic cell a sequence encoding an antigen against which an immune response is desired. Delivery can be achieved by contacting the dendritic cells with a virosome ex vivo, whereupon the infected dendritic cells are provided to the patient. At other times, delivery may be achieved by delivering the virus to the subject so as to infect the dendritic cells in vivo. The dendritic cells then stimulate antigen-specific T cells or B cells in the patient to induce cellular and humoral immune responses to the expressed antigen. In this manner, a patient suffering from a disease or disorder is treated by generating immune cells with the desired specificity.

Any antigen associated with a disease or condition can be delivered to a dendritic cell using a virion as described herein. Identifying antigens associated with a disease or disorder for use in preparing dendritic cell-targeted viral particles. Antigens associated with a number of diseases and conditions are well known in the art. The antigen may be previously known to be associated with a disease or disorder, or may be identified by any method known in the art. For example, antigens of the type of cancer from which the patient is suffering (such as tumor-associated antigens) may be known or may be identified from the tumor itself by any of a variety of methods known in the art.

Tumor-associated antigens are known for a variety of cancers including, for example, renal cell carcinoma, prostate cancer, melanoma, and breast cancer. For example, in some breast cancers, Her-2 receptors are overexpressed on the surface of cancerous cells. Exemplary tumor antigens include, but are not limited to, MAGE, BAGE, RAGE, and NY-ESO-1, which are immunologically privileged regions of the testis (immune-privilegedarea) and unmutated antigens expressed in a variety of tumor cells; lineage specific tumor antigens such as melanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase related proteins, renal cell carcinoma-5T 4, SM22- α, carbonic anhydrase I and IX (also known as G250), hypoxia inducible factors (e.g., HIF-1 α and HIF-2 α), VEGF or Prostate Specific Membrane Antigen (PSMA), Prostate Specific Antigen (PSA), prostate acid phosphate, and six transmembrane epithelial antigen of the prostate (STEAP) NKX3.1, all expressed in normal and neoplastic cells derived from the same tissue; epitope proteins/peptides derived from mutant genes in tumor cells or genes transcribed at different levels in tumors compared to normal cells, such as telomerase, survivin, mesothelin, mutant ras, bcr/abl rearrangement, Her2/neu, mutant or wild-type P53, cytochrome P4501B 1, and aberrantly expressed intron sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that produce distinct idiotypes in myeloma and B-cell lymphoma; epitope proteins/peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein. A number of Tumor-associated Antigens have been reviewed (see, e.g., Tumor-antibodies conjugated By T-Lymphocytes, "Boon T, Cerottini J C, Vandeneyne B, Vanderburggen P, Vanpel A, Annual Review of immunology 12: 337-365, 1994," A stimulating of human Tumor infected monoclonal Tcells, "Renkvist N, Castelli C, Robbins P F, Parmiani G. cancer immunoimmunoimmunoimmunoimmunoimmunoimmunogenethery 50 (1)3-15 MAR, the entire contents of each reference 2001 being incorporated herein By reference).

The antigen may also be an antigen associated with an infectious disease such as, for example, HIV/AIDS. The antigen can be, for example, gp120(Klimstra, W.B. et al 2003.J Virol 77: 12022-12032; Bernard, K.A. et al 2000.Virology 276: 93-103; Byrnes, A.P. et al 1998.J Virol 72: 7349-7356, the entire contents of each reference being incorporated herein by reference). Other exemplary antigens include, but are not limited to: gag, pol, env, tat, nef and rev (Lieberman, J. et al 1997.AIDS Res Hum Retroviruses 13 (5): 383-.

Examples of viral antigens include, but are not limited to, adenoviral polypeptides, alphaviral polypeptides, caliciviral polypeptides (e.g., caliciviral capsid antigens), coronavirus polypeptides, pestivirus polypeptides, Ebola virus (Ebola virus) polypeptides, enteroviral polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (e.g., hepatitis b nuclear or surface antigens, or hepatitis c virus E1 or E2 glycoprotein, nuclear or non-structural proteins), herpesviral polypeptides (e.g., herpes simplex virus or varicella zoster virus glycoprotein), immunodeficiency virus polypeptides (e.g., human immunodeficiency virus envelope or protease), infectious peritonitis virus polypeptides, influenza virus polypeptides (e.g., influenza a hemagglutinin, neuraminidase or nucleoprotein), leukemia virus polypeptides, Marburg virus (Marburg virus) polypeptides, orthomyxovirus polypeptides, Papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin/neuraminidase), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptides), varicella virus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retroviral polypeptides, and rotavirus polypeptides.

Examples of bacterial antigens include, but are not limited to, Actinomyces (Actinomyces) polypeptides, Bacillus (Bacillus) polypeptides, Bacteroides (Bacteroides) polypeptides, borteobacter (Bordetella) polypeptides, Bartonella (Bartonella) polypeptides, burrella (Borrelia) polypeptides (e.g., Borrelia burgdorferi OspA), Brucella (Brucella) polypeptides, Campylobacter (Campylobacter) polypeptides, carbon dioxide phagemid (Capnocytophaga) polypeptides, Chlamydia (Chlamydia) polypeptides, Clostridium (Clostridium) polypeptides, Corynebacterium (Corynebacterium) polypeptides, cockscomb (Coxiella) polypeptides, dermophilus (Dermatophilus) polypeptides, Enterococcus (Enterococcus) polypeptides, Ehrlichia (Ehrlichia) polypeptides, Escherichia (Escherichia) polypeptides, Bacillus proteins, e.g., Clostridium (Haemophilus) polypeptides, Haemophilus (Haemophilus) polypeptides, e polypeptides, e.g., Haemophilus (Haemophilus) polypeptides Helicobacter (Helicobacter) polypeptides, Klebsiella (Klebsiella) polypeptides, L-shaped bacterial polypeptides, Leptospira (Leptospira) polypeptides, Listeria (Listeria) polypeptides, mycobacterium (mycobacterium) polypeptides, Mycoplasma (Mycoplasma) polypeptides, Neisseria (Neisseria) polypeptides, neokestosoma (neickertia) polypeptides, Nocardia (Nocardia) polypeptides, Pasteurella (Pasteurella) polypeptides, Peptococcus (Peptococcus) polypeptides, Peptostreptococcus (Peptostreptococcus) polypeptides, Pneumococcus (Pneumococcus) polypeptides, Proteus (Proteus) polypeptides, Pseudomonas (Pseudomonas) polypeptides, Rickettsia (Rickettsia) polypeptides, rocardia (rochaem) polypeptides, Staphylococcus (Streptococcus pyogenes) polypeptides Treponema (Treponema) polypeptides and Yersinia (Yersinia) polypeptides (e.g., Yersinia pestis (y.pestis) F1 and V antigen).

Examples of fungal antigens include, but are not limited to, Absidia (Absidia) polypeptides, Acremonium (Acremonium) polypeptides, Alternaria (Alternaria) polypeptides, Aspergillus (Aspergillus) polypeptides, Ranunculus (Basidiobolus) polypeptides, Demodenium (Bipolaris) polypeptides, Blastomyces (Blastomyces) polypeptides, Candida (Candida) polypeptides, Coccidioides (Coccidioides) polypeptides, Conidiobolus (Conidiobolus) polypeptides, Cryptococcus (Cryptococcus) polypeptides, Curvularia (Curvalaria) polypeptides, Epidermophyton (Epidermophyton) polypeptides, Exophycea (Exophiala) polypeptides, Geotrichum (Geotrichum) polypeptides, Histoplasma (Histoplasma) polypeptides, Maliduria (Maredella) polypeptides, Microcoriella (Microcoriella) polypeptides, Penicillium (Penicillium) polypeptides, Microcoriella (Microcorium) polypeptides, Microcoriaria) polypeptides, Microcorium (Microcorium) polypeptides, Microcorium, A single spore bottle mold (Phomopora) polypeptide, a Protophthora (Prototheca) polypeptide, a podophyta (Pseudoallotheca) polypeptide, a Pseudocerotobacter (Pseudocerotococcus) polypeptide, a Pythium (Pythium) polypeptide, a nosema (Rhinospora) polypeptide, a Rhizopus (Rhizopus) polypeptide, a Phemonium (Rhizopus) polypeptide, a Phlebia (Scolobasidium) polypeptide, a Mycobacter (Sporothrix) polypeptide, a Staphynopsis (Stemphylium) polypeptide, a Trichophyton (Trichophyton) polypeptide, a Trichophyton (Trichosporon) polypeptide, and a Xylomyces (Xylohyy) polypeptide.

Examples of protozoan parasite antigens include, but are not limited to, Babesia (Babesia) polypeptides, Balantidian (Balattidium) polypeptides, Benzoia (Besnotia) polypeptides, Cryptosporidium (Cryptosporidium) polypeptides, Eimeria (Eimeria) polypeptides, Microsporum encephalitis (Encephalitozoon) polypeptides, Endomonaea (Entamoeba) polypeptides, Giardia (Giardia) polypeptides, Hammondii (Hammondia) polypeptides, Haemophilus (Heptozoon) polypeptides, Isospora (Isospora) polypeptides, Leishmania (LeishCSP) polypeptides, Microsporidia (Microsporidia) polypeptides, Neospora (Neosporira) polypeptides, Agrocybe (Noseola) polypeptides, Pentaphora (pentatricomyces) polypeptides, Pfporaria (Pfagopyrium) polypeptides, Pfsciparum surface protein (Pfagopyrium) proteins, Pfsciparum protein (Pfsciparum protein), Plasmodium protein (Pfsp) proteins, Pfspinorula (SSP-derived protein (Pfavicia) proteins, Pfavicia protein (P-25. sp., Pneumocystis (Pneumocystis) polypeptides, Sarcocystis (Sarcocystis) polypeptides, Schistosoma (Schistosoma) polypeptides, Theileria (Theileria) polypeptides, Toxoplasma (Toxoplasma) polypeptides and Trypanosoma (Trypanosoma) polypeptides.

Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurosttrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunosttomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperina polypeptides, Crenosoma polypeptides, Dictyocarpus polypeptides, Dioctophytome polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Dipart cecum polypeptides, Diplodia, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oedoophora polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascarris polypeptides, Physcoptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometera polypeptides, Stephanosparia polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides.

Examples of ectoparasite antigens include, but are not limited to, polypeptides (including protective antigens and allergens) from the following species: fleas; ticks, including ticks and ticks; flies such as chironomids, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, flies causing myiasis and biting mosquitoes; (ii) ants; a class of spiders; lice species; mites; and stinkbugs, such as bed bugs and kisses.

Once the antigen has been identified and selected, the sequence encoding the desired antigen is identified. Preferably, the sequence comprises cDNA. Following viral infection, the sequence of interest (e.g., a sequence encoding an antigen) is expressed by the target dendritic cell. If contacted ex vivo, the target dendritic cells are then transferred back to the patient, for example by injection, where they interact with immune cells capable of generating an immune response to the desired antigen. In a preferred embodiment, a recombinant virus is injected into the patient, which will transduce the target dendritic cells in situ in the patient. The dendritic cells then express a specific antigen associated with the disease or condition to be treated, and the patient is able to mount an effective immune response to the disease or condition.

The viral vector genome can contain polynucleotide sequences encoding more than one antigen, and upon transduction of a target dendritic cell, an immune response is generated to the plurality of antigens delivered to the cell. In some embodiments, the antigen is associated with a single disease or disorder. In other embodiments, the antigen is associated with a plurality of diseases or disorders.

In some viruses, DC maturation factors that activate and/or stimulate DC maturation are delivered along with the target sequence. In the alternative, the DCs are activated by delivery of DC maturation factors prior to, simultaneously with, or after delivery of the virus. The DC maturation factor can be provided independently by administration of the virus.

As described herein, one or more immunomodulatory or DC maturation factors can be encoded by one or more sequences contained in the viral genome and expressed upon viral infection of dendritic cells. The sequences encoding the immune modulatory factors can also be provided in separate vectors co-transfected with viral vectors encoding one or more antigens in a packaging cell line.

The methods described herein may be used for adoptive immunotherapy in a patient. As described above, antigens against which an immune response is desired are identified. Polynucleotides encoding the desired antigens are obtained and packaged in recombinant viruses. The target dendritic cells are obtained from the patient and transduced with a recombinant virus containing a polynucleotide encoding the desired antigen. The dendritic cells are then transferred back into the patient.

The virions can be injected in vivo, where the virions infect the DC and deliver a target sequence that typically encodes an antigen, the amount of virions is at least 3 × 10⁶IU and may be at least 1 × 10⁷IU, at least 3 × 10⁷IU, at least 1 × 10⁸IU, at least 3 × 10⁸IU, at least 1 × 10⁹IU or at least 3 × 10⁹IU. At selected intervals, DCs from the recipient lymphoid organs can be used to measure expression, for example, by observing marker (such as GFP or luciferase) expression. Nucleic acid monitoring techniques and measurement of Reverse Transcriptase (RT) activity can also be used to analyze the biodistribution of viral particles. Measurable vectors from lentivirusesMagnitude and persistence of response of peripheral blood mononuclear cells, lymph nodes, spleen, or T cells of malignant or target pathogen infected tissue of a recipient of a somatic therapy in response to antigen stimulation. Tissue cells other than DCs, such as epithelial cells and lymphoid cells, can be analyzed for specificity of gene delivery in vivo.

It is widely accepted that the most effective possible method of terminating the AIDS epidemic (and other viral diseases) is an effective prophylactic vaccine. To date, vaccination methods for HIV have not successfully passed phase III trials. Therefore, new effective vaccination strategies are urgently needed. One strategy is vaccination of DCs. In such embodiments, sequences encoding viral proteins (such as those described above) are cloned into a viral vector. The patient is infected with a virus constructed as described herein. In an animal model, molecularly cloned HIV reporter viruses (NFNSZ-r-HSAS, NL-r-HSAS) and clinical isolates can be used to prime animals by tail vein injection. Signs of infection in splenocytes, lymph nodes and peripheral blood can be monitored over time. PCR amplification for HIV-gag protein and flow cytometry for HAS in reporter viruses can be used to test viral integration and replication. Productive in situ DC vaccination may increase resistance to HIV challenge.

Vaccines often include an adjuvant. The lentiviral vector particles described herein can also be administered together with an adjuvant. The adjuvant may be administered with the recombinant virion, before the recombinant virion or after the recombinant virion. If administered with a virion, the ideal adjuvant does not significantly disrupt the integrity of the virion, such as the viral membrane containing the envelope glycoprotein.

Various adjuvants may be used in combination with the virus in order to elicit an immune response to the antigen encoded in the viral vector genome. Preferred adjuvants augment the intrinsic response to the antigen without causing conformational changes in the antigen that affect the qualitative form of the response. Preferred adjuvants include alum, 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211). QS21 is a triterpene glucoside or saponin isolated from The bark of The Quillaja Saponaria Molina tree found in south America (see Kensil et al, Vaccine Design: The Subunit and Adjuvant Aproach (edited by Powell and Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with an immunostimulant such as monophosphoryl lipid a (see Stoute et al, n.engl.j.med.336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today, Nov.15, 1998). Alternatively, a β may be coupled to an adjuvant. For example, as described for vaccination with hepatitis B antigen, the lipopeptide form of A.beta.can be prepared by coupling palmitic acid or other lipids directly to the N-terminus of A.beta. (Livingston, J.Immunol.159, 1383-1392 (1997)). However, such coupling does not substantially alter the conformation of a β so as to affect the nature of the immune response thereto. The adjuvant may be administered as a component of the therapeutic composition with the active agent, or may be administered independently, prior to, concurrently with, or after administration of the therapeutic agent.

One class of adjuvants is aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants may be used with or without other specific immunostimulants such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids (such as polyglutamic acid or polylysine). Another class of adjuvants are oil-in-water emulsion formulations. Such adjuvants may be used with or without other specific immunostimulants such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoleucyl-L-alanine-2- (1 '-2' dipalmitoyl-sn-glycerate-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), N-acetylglutamyl-N-acetylmuramyl-L-AI-D-isoglutamate-L-Ala- Dipalmitoyloxypropionamide (DTP-DPP) theramide. tm.) or other bacterial cell wall components. The oil-in-water emulsion comprises (a) MF59(WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE)) Formulated as submicron particles using a Microfluidics machine (Microfluidics, Newton Mass.) such as a 110Y type Microfluidics machine, (b) SAF containing 10% squalene, 0.4% tween 80, 5% pluronic block copolymer L121 and thr-MDP, which is microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion, and (c) Ribi Adjuvant System (RAS) (Ribi immunolochem, Hamilton, Mont.) containing 2% squalene, 0.2% tween 80 and a mixture of a mixture from the group consisting of monophosphoryl lipid a (MPL), Trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS) (preferably MPL + CWS (Detox + CWS))^TM) ) one or more bacterial cell wall components of the group. Another class of preferred adjuvants are saponin adjuvants such as stimulon. tm. (QS21, Aquila, Worcester, Mass.), or particles produced therefrom such as ISCOMs (immune stimulating complexes) and ISCOMATRIX. Other adjuvants include Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA).

Other adjuvants include cytokines such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF).

Another adjuvant that may be used with the compositions herein is identified by formula (I):

wherein moieties a1 and a2 are independently selected from the group of hydrogen, phosphate and phosphate. Sodium and potassium are exemplary counter ions for phosphate. Moiety R¹、R²、R³、R⁴、R⁵And R⁶Independently selected from 3 to 23 carbons (from C)₃-C₂₃Representative) of the alkyl group. For added clarity, it is noted that when a portion is "independently selected from" a defined group having a plurality of members, it is understood that the selection of a member for a first portion does not affect or limit the selection of a member selected for a second portion in any way. And R¹、R³、R⁵And R⁶The carbon atoms to which they are attached are all asymmetric, so that the carbon atomThe subunits may exist in either R or S stereochemical form. In one embodiment, all of those carbon atoms are in the R stereochemical form, and in another embodiment, all of those carbon atoms are in the S stereochemical form.

"hydrocarbyl" refers to a chemical moiety formed entirely of hydrogen and carbon, in which case the arrangement of carbon atoms may be straight or branched chain, acyclic or cyclic, and the bonds between adjacent carbon atoms may all be single bonds, i.e., to provide a saturated hydrocarbyl group, or there may be double or triple bonds between any two adjacent carbon atoms, i.e., to provide an unsaturated hydrocarbyl group, and the number of carbon atoms in the hydrocarbyl group is between 3 and 24 carbon atoms. The hydrocarbyl group can be alkyl, where representative straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, including undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, and the like; and branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic hydrocarbyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; and the unsaturated cyclic hydrocarbon group includes cyclopentenyl and cyclohexenyl and the like. Unsaturated hydrocarbyl groups contain at least one double or triple bond between adjacent carbon atoms (referred to as "alkenyl" or "alkynyl", respectively, if the hydrocarbyl group is a non-cyclic hydrocarbyl group, and as cycloalkenyl and cycloalkynyl, respectively, if the hydrocarbyl group is an at least partially cyclic hydrocarbyl group). Representative straight and branched chain alkenyls include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl, and the like; and representative straight and branched alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-butynyl, and the like.

Adjuvants of formula (I) may be obtained by synthetic methods known in the art, for example, by the synthetic methods disclosed in PCT international publication No. WO 2009/035528, which is incorporated herein by reference, and the publications identified in WO 2009/035528, each of which is also incorporated herein by reference. Certain adjuvants are also commercially available. A preferred adjuvant is product No. 699800, identified in the Avanti Polar Lipids catalog of Alabaster AL, see E1 in combination with E10, infra.

In various embodiments of the invention, the adjuvant has the chemical structure of formula (I), but moieties a1, a2, R1, R2, R3, R4, R5 and R6 are selected from a subset of the options previously provided for these moieties, wherein these subsets are identified below by E1, E2, and the like.

E1：A₁Is a phosphate or a phosphonate and a2 is hydrogen.

E2：R¹、R³、R⁵And R⁶Is C₃-C₂₁An alkyl group; and R is²And R⁴Is C₅-C₂₃A hydrocarbyl group.

E3：R¹、R³、R⁵And R⁶Is C⁵-C¹⁷An alkyl group; and R is²And R⁴Is C⁷-C¹⁹A hydrocarbyl group.

E4：R¹、R³、R⁵And R⁶Is C₇-C₁₅An alkyl group; and R is²And R⁴Is C₉-C₁₇A hydrocarbyl group.

E5：R¹、R³、R⁵And R⁶Is C₉-C₁₃An alkyl group; and R is²And R⁴Is C₁₁-C₁₅A hydrocarbyl group.

E6：R¹、R³、R⁵And R⁶Is C₉-C₁₅An alkyl group; and R is²And R⁴Is C₁₁-C₁₇A hydrocarbyl group.

E7：R¹、R³、R⁵And R⁶Is C₇-C₁₃An alkyl group; and R is²And R⁴Is C₉-C₁₅A hydrocarbyl group.

E8：R¹、R³、R⁵And R⁶Is C₁₁-C₂₀An alkyl group; and R is²And R⁴Is C₁₂-C₂₀A hydrocarbyl group.

E9：R¹、R³、R⁵And R⁶Is C₁₁An alkyl group; and R is²And R⁴Is C₁₃A hydrocarbyl group.

E10：R¹、R³、R⁵And R⁶Is undecyl and R²And R⁴Is tridecyl.

In certain options, each of E2 to E10 is in combination with embodiment E1, and/or the hydrocarbyl group of E2 to E9 is an alkyl group, preferably a straight-chain alkyl group.

The adjuvant of formula (I) may optionally be formulated into a pharmaceutical composition with a co-adjuvant, each as discussed below. In this regard, reference is made to U.S. patent publication No. 2008/0131466, which provides, for example, aqueous solution formulations (AF) and stable emulsion formulations (SE) of GLA adjuvants, wherein these formulations can be used with any adjuvant of formula (I).

Adjuvants may be administered with the viruses of the present invention as a single composition, or may be administered prior to, simultaneously with, or after administration of the recombinant viruses of the present invention. The immunogen and adjuvant may be packaged and supplied in the same vial, or may be packaged in separate vials and mixed prior to use. Immunogens and adjuvants are typically packaged with labels indicating the intended therapeutic application. If the immunogen and adjuvant are packaged separately, the package will typically include instructions for mixing prior to use. The choice of adjuvant and/or carrier depends on the stability of the vaccine comprising the adjuvant, the route of administration, the administration schedule, the efficacy of the adjuvant on the species to be vaccinated, and in humans, a pharmaceutically acceptable adjuvant is one that has been approved by the relevant regulatory authorities or is suitable for human administration. For example, Freund's complete adjuvant is not suitable for human administration. Alum, MPL and QS21 are preferred. Optionally, two or more different adjuvants may be used simultaneously, such as alum with MPL, alum with QS21, MPL with QS21, and alum, QS21 and MPL. In addition, Freund's incomplete adjuvant (Chang et al, Advanced Drug Delivery Reviews 32, 173-186(1998)) may be used, optionally in combination with any one of alum, QS21, and MPL, and all combinations thereof.

E. Pharmaceutical composition and kit

Also encompassed herein are pharmaceutical compositions and kits containing the viruses provided herein and one or more components. The pharmaceutical composition may comprise a viral vector particle and a pharmaceutical carrier as provided herein. A kit can include a pharmaceutical composition and/or combination provided herein, as well as one or more components, such as instructions for use, a device for administering the compound to a subject, and a device for administering the compound to a subject.

Provided herein are pharmaceutical compositions containing viral particles as provided herein and a suitable pharmaceutical carrier. The pharmaceutical compositions provided herein can be in various forms, for example, can be in solid, liquid, powder, aqueous solution, or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and/or additives can be formulated by conventional methods and can be administered to a subject in a suitable dosage. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can protect the composition from degradation in vivo.

The viral vector particles provided herein can be packaged as a kit. Kits may optionally include one or more components, such as instructions for use, devices, and additional reagents, as well as components for practicing the methods of the invention, such as tubes, containers, and syringes. An exemplary kit can include a virus provided herein, and can optionally include instructions for use, a device for detecting the virus in a subject, a device for administering the virus to the subject, and a device for administering the compound to the subject.

Also encompassed herein are kits comprising a polynucleotide encoding a gene of interest, typically an antigen. The kit may include at least one plasmid encoding viral packaging components and a vector encoding a sindbis virus E2 glycoprotein variant. Some kits will contain at least one plasmid encoding viral packaging components, a vector encoding sindbis virus E2 glycoprotein variant, and a vector encoding at least one DC maturation factor.

Also encompassed herein are kits comprising a viral vector encoding a sequence of interest (typically an antigen) and optionally a polynucleotide sequence encoding a DC maturation factor. In some kits, the kit includes at least one plasmid encoding a viral packaging component and a vector encoding a sindbis virus E2 glycoprotein variant.

The kit may also contain instructions. The specification generally includes tangible expressions that describe: the virus and optional other components contained in the kit, as well as methods of administration, including methods for determining the appropriate state of a subject, the appropriate amount to administer, and the appropriate method of administration for administering the virus. The instructions may also include instructions for monitoring the subject over the duration of the treatment period.

The kits provided herein can also include a device for administering the virus to a subject. Any of a variety of devices known in the art for administering drugs or vaccines can be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eye droppers. Generally, the device used to administer the virus of the kit should be compatible with the virus of the kit; for example, a needle-free injection device (such as a high pressure injection device) may be included in a kit, but typically will not be included in a kit, where viruses are not damaged by high pressure injection.

The kits provided herein can also include a device for administering a compound (such as a DC activator or stimulator) to a subject. Any of a variety of devices known in the art for administering a drug to a subject can be included in the kits provided herein. Exemplary devices include hypodermic needles, intravenous needles, catheters, needle-free injection devices, but are not limited to hypodermic needles, intravenous needles, catheters, needle-free injection devices, inhalers, and liquid dispensers, such as eye droppers. Generally, the device used to administer the compounds of the kit should be compatible with the desired method of administering the compounds.

The following examples are provided by way of illustration and not by way of limitation.

Examples

Example 1

Engineering of Sindbis virus envelope variants

Sindbis Virus (SV), a member of the alphavirus genus and Chlamydomonas family, is capable of infecting DCs, possibly via DC-SIGN (Klimstra, W.B. et al 2003. J.Virol.77: 12022-12032, the entire contents of which are incorporated herein by reference). However, typical viral receptors for laboratory strains of SV are cell surface Heparan Sulfate (HS) found in many cell types (Strauss, J.H. et al 01994. Arch.Virol.9: 473-484; Byrnes, A.P. and D.E.Griffin.1998.J.Virol.72: 7349-7356, the entire contents of each reference being incorporated herein by reference). In an attempt to reduce heparan sulfate binding, Wang et al constructed a mutant E2 envelope (called SVGmu) (US 2008/0019998, incorporated in its entirety). Part of their strategy involved deletion of four amino acids at the junction of the E3/E2 proprotein and deletion of two amino acids followed by addition of a 10 amino acid sequence from hemagglutinin (see fig. 1A and 1B). The resulting E2 protein was expressed as a fusion of E3 and E2 (also known as pE2) because the native cleavage sequence was disrupted and the resulting E2 protein displayed a foreign epitope (hemagglutinin). As shown below, SVGmu has, inter alia, the problem of low expression levels.

The inventors have used different strategies to engineer the E2 glycoprotein of sindbis virus to reduce binding to heparan sulfate, increase specificity for dendritic cells and increase expression. The general approach to achieve these features is to increase the infectivity of dendritic cells by changing residue 160 of E2 (residue 233 in fig. 1) to a non-acidic amino acid, especially an amino acid other than alanine, or deleting it, to decrease heparin binding by reducing the net positive charge of the protein, to remove the HA (hemagglutinin) epitope and to restore the cleavage site at the N-terminus of E2 (in this case the furin cleavage site). As part of the viral envelope, these E2 glycoproteins are able to mediate infection of DCs as well as reduce or eliminate infection of other cell types.

Following these principles, several variant sequences of E2 were designed and shown in the table below. Bold font indicates changes in the envelope sequence (gene bank NC 001547.1) of sindbis virus by HR strain.

Nucleic acid sequences encoding some of the variants were synthetic (see also fig. 1), including nucleic acid sequences optimized for codons in humans. The DNA of the variant is cloned into an expression vector, such as pcDNA 3.

Example 2

Preparation of viral vector particles comprising variants of the envelope glycoprotein of Sindbis Virus E2

Viruses pseudotyped with sindbis envelope were prepared by standard calcium phosphate-mediated transient transfection of 293T cells with viral vectors (such as FUGW) or derivatives thereof, packaging plasmids encoding gag, pol and rev, and variant sindbis virus envelope sequences. FUGW is a self-inactivating lentiviral vector carrying the human ubiquitin-C promoter driving expression of the GFP gene (Lois, C et al 2002: Science 295: 868-872, which is incorporated herein by reference in its entirety). Lentiviral transfer vectors (FUGW and derivatives thereof) are third generation HIV-based lentiviral vectors (see generally, Cockrell and KafriMol. Biotechnol.36: 184, 2007) in which most of the U3 region of the 3'LTR is deleted, resulting from inactivation of the 3' -LTR.

The preparation of recombinant lentiviral vectors was achieved by calcium phosphate (CaPO4) mediated transient transfection of 293T CELLs (293LTV CELL line; CELL BIOLABS INC, LTV-100). 293T cells were transfected with four plasmids precipitated with CaPO 4. The following four plasmids were used to prepare the lentiviral vector formulations and are shown schematically in figure 3 and correspond to the following: i) a lentiviral vector; ii) a plasmid encoding HIV Rev; iii) a plasmid encoding HIV Gag/Pol; and iv) a plasmid encoding the envelope. Lentiviral vectors can encode desired antigens or immune regulatory elements and contain specifically targeted deletions to prevent integration into the host chromosome of infected cells. Other examples of surrogate plasmids that can be used for transient transfection include those that encode the polymerase holoenzyme, which carries a mutation in its integrase that makes it defective, such as the D64V mutation described herein. For certain purposes, the envelope-encoding plasmid can encode a non-DC-targeted pantropic envelope, such as VSV-G.

For the experiments described herein, the CaPO4 pellet containing 120 μ g vector and 60 μ g each of Gag/Pol and Rev plasmids and 240 μ g of envelope plasmid was filtered through a 0.45 μm pore size filter and added to approximately 6 × 10mL of DMEM medium grown in spinner flasks and containing 75mL of fetal bovine serum supplemented with 10% fetal bovine serum⁷In 293T cells 6 hours post-transfection, media was replaced with 100mL fresh media and collected 36 hours post-transfection, culture supernatant was centrifuged at low speed (1200rpm) to pellet cell debris followed by filtration through a 0.45 μm filter the filtrate containing the lentiviral vector preparation was concentrated by centrifugation at 17,700 × g for 5 hours, optionally at 20 deg.C, then the precipitated lentiviral vector was resuspended in PBS at the desired volume, this method typically produced ≧ 5 × 10 when using the Sindbis viral glycoprotein envelope described herein⁵IU/mL, or a total of 5 × 10 for each spinner flask culture⁷IU. more typically this method results in a total of at least 1 × 10 for each spinner culture⁸IU。

In more detail, on day 4, 150mL of cell culture medium is added to the spinner flask (RB), the spinner flask is placed in a 37 ℃ roller frame incubator (0.2rpm) for about 1 hour, confluent 15cm dishes are delivered to each RB, on day 2, medium is aspirated from the RB and replaced with 100mL of pre-heated medium, on day 1, the cells are seeded for use as transfection, the RB is pre-heated with 100mL of cell culture medium for about 1 hour, medium is aspirated and 10-12mL of PBS is added, RB is side-placed and spun two times to coat the cells, PBS is aspirated and 10-12mL of trypsin solution is added, RB is side-placed and spun two times to coat the cells again, trypsin solution is aspirated and placed in the incubator for 5min, 10mL of warm medium is added to the RB, and RB is vigorously spun one time to separate the cells, using a 10mL pipette, the cells are moved up and down (e.g.) 10 times to ensure a single cell suspension is moved to a new container and the RB is diluted with medium (3540. each RB is counted as 857. each cell culture medium and cell is counted with 8584. each cell count 10. on day 3.7. day, cell culture medium is added to coat cells, cell culture medium⁷Perml was inoculated into RB and kept in the incubator overnight.

On day 0, approximately 22 hours after inoculation, plasmid solutions were prepared as follows. For each RB, plasmid solutions (120. mu.g vector, 60. mu.g Gag/Pol, 60. mu.g Rev, 60. mu.g envelope), 2.5mL of 1.25M CaCl2, and filtered sterile water were mixed together to a final volume of 12.5 mL. To 50ml tubes (1 tube/RB) were added 2.7ml of 2 XHBS (50mM HEPES, 10mM KCl, 12mM glucose, 280mM NaCl, 1.5mM Na2HPO4-7H2O, pH 7.0, sterile filtered) buffer.

12.7mL of the water-CaCl 2-DNA mixture was added dropwise to 12.7mL of 2 XHBS while vortexing at the media setting. The tube was capped and vortexing (maximum speed) continued for 5-10 seconds. 25ml of medium was removed from the RB and the pellet (25ml) was added to the RB. After 6 hours of incubation, the medium was aspirated and 100ml of fresh, pre-warmed cell culture medium was added. Placed back in the 37 ° incubator.

36 to 48 hours post-transfection, the supernatant from RB was collected into 250mL conical tubes and processed as follows. The supernatant was centrifuged at 2000rpm for 10 min. The supernatant was filtered through a 0.45um filter. The supernatant in the 500ml tube was centrifuged at 10,000rpm for 5 hours at 20 ℃. The vector was resuspended in PBS or HBSS to the desired concentration and stored at-80 ℃.

The pseudotyped viral vectors thus obtained are hereinafter referred to as FUGWA/1, FUGWA/2, etc. The viral vector genome enveloped by the VSV-G glycoprotein is hereinafter referred to as FUGWA/SV-G.

Example 3

Preparation of lentiviral vector particles comprising Sindbis Virus envelope glycoprotein

In this example, the titer of lentiviral vectors pseudotyped with different Sindbis virus envelopes was determined. The E2 glycoproteins used were SIN-Var1(SEQ ID No.3), SIN-Var2(SEQ ID No.4), SIN-Var3(SEQ ID No.5), SVGMu (SEQ ID No.2), HR (SEQ ID No. 18).

Lentiviral vector particles pseudotyped with sindbis virus glycoprotein were generated by transfected 293T cells as described in example 2. Crude supernatants were harvested 48 hours post transfection and used to transduce 293T cells expressing human DC-SIGN (293-DCSIGN), which had been seeded at 2E5 cells/well in 6-well dishes the previous day. After incubation at 37 ℃ for 72 hours, titers were determined by analyzing transduced cells on a Guava Easy-Cyte cell counter (Millipore). A total of 25,000 events were counted to determine the percentage of GFP + transduced cells, which was then used to calculate the GFP titer (IU, infectious units) for each virus.

To facilitate the study of targeted transduction, cell lines expressing human DC-SIGN were constructed. The cell line was generated by stably transducing parental 293T cells with a VSVG-pseudotyped lentiviral vector containing the coding sequence for human DC-SIGN. The cDNA of human DC-SIGN was amplified from plasmid pUNO-hDCSIGN1Aa (InvivoGene) and cloned downstream of the human ubiquitin-C promoter in lentiviral plasmid FUW to generate FUW-hDCSIGN. Alternatively, cell lines were generated by stably transducing parental 293T cells with VSVG-pseudotyped facultative (non-lentiviral) vectors containing the coding sequence of DC-SIGN to better facilitate downstream nucleic acid-based analysis of lentiviral vector particle transduction. The lentiviral vector or facultative vector was then pseudotyped with VSVG and used to transduce 293T cells. Alternatively, cell lines were generated by stably transducing parental 293T cells with a plasmid encoding human DC-SIGN. The resulting cells were subjected to antibody staining (anti-human DC-SIGN antibodies (BD Biosciences)) and cell sorting to generate a homogeneous population of DC-SIGN + cell lines.

In three independent experiments, the titer of lentiviral vectors pseudotyped with the SIN-Var1 envelope was about 10 times higher than those pseudotyped with SVGMu or pseudotyped with the HR strain of Sindbis virus. In subsequent studies, the productivity of three sindbis envelope variants were compared. Representative results are shown. The Sin-Var1, Sin-Var2, and SIN-Var3 envelopes produced lentiviral vector particles with similar overall titers.

The specificity of viral vector particles comprising sindbis virus variant E2 glycoprotein was assessed by transducing 293T.

Target cells (293T. hDC-SIGN or 293T cells) were seeded in 24-well plates (0.2 × 10 per well)⁶Individual cells) and transduced with viral supernatant (1 ml per well) by centrifuging the culture dish at 2,500rpm for 90min at 30 ℃. Subsequently, the supernatant was replaced with fresh medium and incubated at 37 ℃ for 3 days. The percentage of cells expressing the marker was measured by flow cytometry.

As shown in the table below, E2 variant 1 and E2 variant 3 preferentially target cells expressing hDC-SIGN (specific to the cells).

Example 4

Immunogenicity of lentiviral vector particles comprising sindbis virus envelope glycoprotein

In this example, the immunogenicity of lentiviral vectors pseudotyped with different Sindbis viral envelopes was evaluated. More specifically, the amount of antigen-specific CD8T cells and their cytokine secretion profile were determined. The E2 glycoproteins used were SIN-Var1(SEQ ID No.3), SIN-Var2(SEQ ID No.4), SIN-Var3(SEQ ID No.5), SVGMu (SEQ ID No. 2).

The viral genome comprises a sequence encoding Ovalbumin (OVA). Lentiviral vector particles were generated by transfection of 293T cells as described in example 2. The supernatant was collected and the amount of p24 was determined using an ELISA kit (Advanced Bioscience Labs, Kensington MD). Protein p24 is the product of the HIV nucleoprotein and gag genes found in pseudotyped virions. C57BL/6 mice (5 mice per group) were immunized subcutaneously with integration-deficient lentiviral vectors encoding ovalbumin OVA. On day 9, the number and function of OVA257(SIINFEKL) (SEQ ID NO 24) peptide-specific CD8T cells in the spleen and cytokine secretion profile were determined by MHC-I/peptide multimer and intracellular cytokine staining.

Briefly, spleens were extracted and homogenized by pressing through a 70uM nylon filter, red blood cells were lysed by hypotonic shock by brief exposure to distilled water, and immediately restored to an isotonic environment with the addition of 10 XPBS at 25 deg.C in PBS with 2% FCS and 2mM EDTA (FACS buffer), approximately 5 × 10 Kb/OVA257 pentamer (Prolmmune) for each sample at approximately 5 ×⁶Individual splenocytes were stained. Then, the cells were washed twice and labeled with the feasibility dye LIVE/DEAD Near-isolated (L/D NIR; Invitrogen) and the following fluorescent dyes at 4 deg.C: CD44 FITC, CD19 PerCP-Cy5.5, and CD8 Pacific Blue (eBioscience). Data (50,000 CD8+ events) were collected on a BD LSR II flow cytometer and analyzed with FlowJo software (TreeStar). The gating strategy (gating strategy) for identifying CD8T cells was as follows: lymphocyte (forward scatter lo-med, side scatter lo), single cell (side scatter area ═ side scatter)Height), viable cells (L/D NIR-), CD8T cells (CD8+ CD19-), the percentage of cells expressing IFN- γ within the CD8+ gate was determined and depicted in fig. 4A and 4B the horizontal line depicts the average value the nonspecific IFN- γ staining in spleen cells of vehicle (HBSS) injected mice, which were stimulated with peptide in vitro, all samples not cultured with peptide had less than 0.2% (not shown) IFN- γ staining, in addition to the CD8T cell fraction producing IFN- γ, the IFN- γ + cell fraction producing TNF α and/or IL-2 was also depicted as indicated in the legend.

In one set of experiments, the amount of virus used contained 2500ng or 125ng of p 24. FIG. 4A illustrates that lentiviral vectors pseudotyped with Sindbis variant envelopes have similar in vivo activity. As shown in the left-most panel, the average of antigen-specific CD8T cells from two different doses was essentially the same. In addition, the average percentage of IFN- γ cells was similar. The pattern of cytokine secretion (especially the fraction of IFN-. gamma.positive cells that also express IL-2 or TNF-. alpha.) is similar, with the highest percentage of IFN-. gamma.cells being negative for IL-2 and TNF-. alpha.s.

In another set of experiments, groups of 5 mice received serial dilutions of virus or vehicle. Viruses were pseudotyped with SinVarl or SVGMu. The percentage of cells with the CD44hi H-2Kb/OVA257 pentamer + phenotype is depicted in FIG. 4B. The connecting lines depict the average values. As shown in fig. 4B, SinVarl-pseudotyped LVs induced substantially greater expansion of antigen-specific CD8T cells than SVGmu. Furthermore, SinVarl-pseudotyped lentiviral vectors induced a greater functional CD8T cell response than SVGmu (fig. 4C). Nonspecific IFN- γ staining was determined in HBSS-injected (vehicle) mice re-stimulated with peptide. All samples not incubated with peptide had less than 0.2% IFN-. gamma.staining (not shown).

Example 5

Construction of non-Integrated Lentiviral vectors (NILV)

A plurality of non-integrative lentivirus vectors are constructed. FIG. 5A shows a schematic of an exemplary lentiviral vector. The upper panel shows the vector in proviral form. All vectors contain a splice donor, a packaging signal sequence (psi), a Rev Reactive Element (RRE), a splice donor, a splice acceptor, a central polypurine channel (cPPT), and a WPRE element. In addition, all vector constructs contain a promoter for expression in mammalian cells, and a sequence of interest labeled "antigen" in the exemplary construct. Promoters used in the examples include the human ubiquitin-C promoter (UbiC), cytomegalovirus early transient promoter (CMV), and Rous sarcoma virus promoter (RSV).

The enlargement of the region U3 is shown below as an open box with the PPT (polypurine channel) sequence immediately upstream. Three different zones of U3 are shown in schematic form below; their sequences are shown in fig. 5B in SEQ ID NO: 21-23. The construct contained a deletion in the U3 region. The SIN construct has a deletion of about 130 nucleotides in U3 (Miyoshi et al J Virol 72: 8150, 1998; Yu et al PNAS 83: 3194, 1986) which removes the TATA box, thereby eliminating LTR promoter activity. Deletions in constructs 703 and 704 increased expression from lentiviral vectors (Bayer et al Mol Therapy 16: 1968, 2008, incorporated in its entirety). In addition, construct 704 contains a deletion of the 3' PPT, which reduces integration of the vector (WO2009/076524, the entire content of which is incorporated). The sequence of the U3 region for all constructs is shown in fig. 5B. The 3' PPT starts at position 3 and shows an extended deletion relative to the vector lacking SIN.

Example 6

Expression in dendritic cells following transduction with lentiviral vector particles

This example shows GFP (green fluorescent protein) expression levels in dendritic cells generated from vectors with extended U3 deletions.

A series of viruses were prepared by transfecting 293T cells as described above. All viruses comprise the SIN-Var1 envelope, a vector genome containing the UbiC or CMV promoter operably linked to the GFP transgene, and a defective integrase containing the D64V mutation. Crude supernatants were harvested 48 hours after transduction, and equal volumes of each supernatant were used to transduce 293T-DCSIGN cells.

GFP expression was measured 72 hours after transduction using a GUAVAEasy-Cyte flow cytometer. Each transduced cell population was counted for a total of 50,000 events. Data was analyzed using FlowJo cytometric analysis software.

Similar results were obtained with the UbiC (fig. 6, panel a) and CMV (fig. 6, panel B) promoters. Lentiviral vectors (703 and 704) containing the extended U3 deletion showed higher overall transgene expression relative to vectors lacking SIN. The absence of PPT in construct 704 slightly reduced expression relative to construct 703.

Example 7

Vectors with a large deletion in U3 are non-integrative

This example illustrates the relative integration efficiency of SIN-Var1 pseudotyped vectors containing different combinations of vector deletions with defective or wild type integrase after 293-DC-SIGN cell transduction.

Vectors 703 and 704 with the U3 deletion (see FIGS. 5A and 5B), SIN were transfected as above with either wild-type integrase gene or mutant integrase gene, the vectors in these experiments all contained GFP-2A-Neo transgenes encoding GFP and G418 resistance, the reading frames were linked via 2A self-cleaving peptides to produce individual proteins⁵293-DC-SIGN cells seeded at cell/well in 6-well dishes after transduction, cells were treated with trypsin every 72 hours at each passage 2mL of DMEM + 10% FBS 2 × 10⁵The individual cells were then seeded in 6-well plates. Residual cellsTo determine the number of GFP + cells by flow cytometry.

In fig. 7, the relative GFP titers are presented as a fraction of the titers observed at the first passage. Loss of GFP expression reflects loss of the GFP transgene as a result of lack of integration during the initial transduction step. All viruses containing the D64V mutant integrase (IN-) were found to be non-integrating. Furthermore, viruses containing PPT deletions (704) were found to be integration-deficient even IN the presence of the wild-type IN gene. This shows that the deletion of 3' PPT in combination with the integrase mutation provides a redundant safety mechanism.

Example 8

The immunogenicity of the integrative and non-integrative lentivirus vectors are equivalent

In this example, CD8T cell responses were assessed following immunization with either an integrating or non-integrating virus.

Integration type (Int) with Gag antigen encoding Simian Immunodeficiency Virus (SIV)^wt) Or non-integrated (Int)^D64V) Lentiviral vector 2.5 × 10¹⁰Genome groups were immunized subcutaneously in C57BL/6 mice. The number and function of SIV Gag-specific CD8T cells in the spleen was determined by intracellular cytokine staining on day 10 as described above, except for restimulation with SIV Gag-derived peptide AAVKNWMTQTL. FIG. 8 illustrates that non-integrating lentiviral vectors can elicit a CD8T cell response comparable to integrating lentiviral vectors. In addition, the pattern of cytokine expression is similar.

Example 9

Immunization with DC-NILV to provide therapeutic effects

In this example, mice that have received tumor cells are treated by immunization with DC-NILV expressing a tumor antigen.

BALB/c mice were injected subcutaneously with 2 × 10⁴And CT26 colon cancer cell. One day later, mice were treated subcutaneously with vehicle or a formulation of SINvar1 pseudotyped lentiviral vector particles containing 3.2 μ g of p24 capsid protein. The viral vector envelope comprises a variant of Sindbis virus E2 as described above, and the vector is a non-integrating vector and encodes an AH1A5 peptide (SPSYAYHQF; SEQ ID NO.25), a modified MMTV gp70 CD8T cell epitope, a rejection antigen associated with CT26 tumor cells. Initial tumor growth and long-term survival of immunized versus control mice are depicted. Figure 9 shows that tumors grew more slowly and, in addition, survival (measured over 75 days) was substantially better in mice receiving DC-NILV (60% survival compared to 20%). Thus, non-integrating lentiviral vectors targeting DC (DC-NILV) were effective in the therapeutic treatment of tumors.

Embodiments of the present invention include, but are not limited to, the following.

1. A retroviral (e.g., lentiviral) vector particle comprising:

(a) an envelope comprising the amino acid sequence of SEQ ID NO:1, wherein 160X is absent or is an amino acid other than glutamic acid, or a polypeptide of SEQ ID NO:1, which variant is identical to SEQ id no:1 has at least 80% sequence identity; wherein the E2 glycoprotein or variant thereof is not fused to E3; and

(b) a lentivirus genome comprising a sequence of interest.

2. The retroviral vector particle of embodiment 1, wherein the E2 glycoprotein or variant binds to DC-SIGN.

3. The retroviral vector particle of embodiment 1 or embodiment 2, wherein the 160X is absent or is glycine, alanine, valine, leucine or isoleucine, such as selected from glycine, valine, leucine or isoleucine.

4. The retroviral vector particle of embodiment 1 or embodiment 2, wherein 160X is glycine.

5. The retroviral vector particle of embodiment 1,2, 3, or 4, wherein at least one amino acid of the E2 glycoprotein variant is altered to reduce its net positive charge.

6. The retroviral vector particle of embodiment 5, wherein the altered amino acid is selected from the group consisting of SEQ ID NO:1, lysine 70, lysine 76, or lysine 159, and said substitutions are optionally independently selected from glutamic acid or aspartic acid.

7. The retroviral vector particle of embodiment 1,2, 3, 4,5 or 6, wherein the E2 variant sequence is SEQ ID No.2, 3 or 4; (variants 1,2 and 3), optionally comprising one or more further substitutions, insertions or deletions.

8. The retroviral vector particle of embodiment 1,2, 3, 4,5, 6 or 7 which is a variant wherein the nucleotide sequence of SEQ ID NO:1 or has one or two substitutions which do not affect the ability of the variant to infect DCs, but do not alter the number of amino acids in this region.

The present invention encompasses any combination of the above-described embodiments, as set forth in the following non-limiting combinations, wherein ">" represents a reference to a previously numbered embodiment: 4 > 2 > 1; 5 > 4 > 1; 5 is more than 4 and more than 2 is more than 1; 5 > 3 > 1; 5 is more than 3 and more than 2 is more than 1; 6 is more than 5 and more than 4 is more than 1; 6 is more than 5 and more than 4 and more than 2 and more than 1; 6 is more than 5 and more than 3 and more than 2 and more than 1; 6 is more than 4 and more than 2 is more than 1; 6 is more than 3 and more than 2 is more than 1; 6 is more than 5 and more than 3 is more than 1; 7 is more than 6 and more than 5 and more than 4 and more than 1; 7 is more than 6 and more than 5 and more than 4 and more than 2 and more than 1; 7 is more than 6 and more than 5 and more than 3 and more than 1; 7 is more than 6 and more than 5 and more than 3 and more than 2 and more than 1; 7 is more than 6 and more than 4 and more than 2 and more than 1; 7 is more than 5 and more than 4 is more than 1; 7 is more than 5 and more than 4 and more than 2 and more than 1; 7 is more than 5 and more than 3 is more than 1; 7 is more than 5 and more than 3 and more than 2 and more than 1; 7 is more than 4 and more than 2 and more than 1; 7 is more than 3 and more than 2 is more than 1; 7 is more than 6 and more than 4 is more than 1; 7 is more than 6 and more than 4 and more than 2 and more than 1; 7 is more than 6 and more than 3 is more than 1; 7 is more than 6 and more than 3 and more than 2 and more than 1; 7 is more than 6 and more than 2 is more than 1; 8 is more than 4 and more than 2 and more than 1; 8 is more than 5 and more than 4 is more than 1; 8 is more than 5 and more than 4 and more than 2 and more than 1; 8 is more than 5 and more than 3 is more than 1; 8 is more than 5 and more than 3 and more than 2 and more than 1; 8 is more than 6 and more than 5 and more than 4 and more than 1; 8 is more than 6 and more than 5 and more than 4 and more than 2 and more than 1; 8 is more than 6, more than 5, more than 3, more than 2 and more than 1; 8 is more than 6 and more than 4 and more than 2 and more than 1; 8 is more than 6 and more than 3 and more than 2 and more than 1; 8 is more than 6 and more than 5 and more than 3 and more than 1; 8 is more than 7 and more than 6 and more than 5 and more than 4 and more than 1; 8 is more than 7 and more than 6 and more than 5 and more than 4 and more than 2 and more than 1; 8 is more than 7 and more than 6 and more than 5 and more than 3 and more than 1; 8 is more than 7, more than 6, more than 5, more than 3, more than 2 and more than 1; 8 is more than 7 and more than 6 and more than 4 and more than 2 and more than 1; 8 is more than 7 and more than 5 and more than 4 and more than 1; 8 is more than 7 and more than 5 and more than 4 and more than 2 and more than 1; 8 is more than 7 and more than 5 and more than 3 and more than 1; 8 is more than 7, more than 5, more than 3, more than 2 and more than 1; 8 is more than 7 and more than 4 and more than 2 and more than 1; 8 is more than 7 and more than 3 and more than 2 and more than 1; 8 is more than 7 and more than 6 and more than 4 and more than 1; 8 is more than 7 and more than 6 and more than 3 and more than 1; 8 is more than 7, more than 6, more than 3, more than 2 and more than 1; 8 is more than 7 and more than 6 and more than 2 and more than 1.

Other embodiments of the invention include:

9. the retroviral vector particle of any one of the above embodiments, wherein the target sequence encodes a tumor specific antigen or a virus derived antigen, such as an HIV or SIV antigen.

10. A retroviral (e.g., lentiviral) vector genome pseudotyped in an envelope comprising a Sindbis virus E2 glycoprotein variant, the genome comprising a sequence of interest,

wherein the E2 glycoprotein variant facilitates infection of dendritic cells by the retroviral vector particle; and wherein the E2 glycoprotein variant has an amino acid sequence as defined in any one of embodiments 1 to 9 above.

11. A retroviral vector packaging system for producing a pseudotyped retroviral vector particle comprising:

(i) a first nucleic acid molecule encoding the polypeptide of SEQ ID NO:1, wherein 160X is absent or is an amino acid other than glutamic acid, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, said variant being identical to SEQ ID NO:1 has at least 80% sequence identity; and

(ii) a second nucleic acid molecule, wherein said second nucleic acid molecule can be transcribed and the transcript can be assembled into a pseudotyped retroviral vector particle.

12. The packaging system of embodiment 11, wherein the E2 glycoprotein has an amino acid sequence as defined in any one of embodiments 1 to 9 above.

13. The packaging system of embodiment 11 or 12, wherein the first nucleic acid molecule encodes E3/E2 glycoprotein, optionally in the form of sindbis virus E3/E2/6K/E1 polyprotein.

14. The packaging system of embodiment 13, wherein the E3 sequence corresponds to SEQ ID NO: 20 or a variant thereof which is substantially identical to SEQ ID NO: 20, wherein residues 62-65 are RSKR (SEQ ID NO: 27) and the variant is capable of being incorporated into a pseudotyped viral envelope, optionally further wherein residue 1 of the E2 polyprotein is Ser.

15. The packaging system of embodiments 11, 12, 13, or 14, further comprising a third nucleic acid molecule encoding gag and pol proteins.

16. The packaging system of any one of embodiments 11-15, wherein the second nucleic acid molecule comprises a sequence of interest.

17. A cell transfected with a first nucleic acid molecule and a second nucleic acid molecule according to any one of embodiments 11 to 16.

18. An isolated nucleic acid molecule encoding a protein according to any one of embodiments 1 to 9 or the E3/E2 glycoprotein as defined in embodiment 13 or 14.

19. An expression vector comprising the nucleic acid molecule of embodiment 18.

20. A host cell comprising the expression vector of embodiment 19.

21. A method for preparing the retroviral vector particle of any one of embodiments 1 to 10 comprising expressing in a cell

(i) A first nucleic acid molecule encoding the polypeptide of SEQ ID NO:1, wherein 160X is other than glutamic acid, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, said variant being substantially identical to SEQ id no:1 has at least 80% sequence identity; and

(ii) a second nucleic acid molecule, wherein said second nucleic acid molecule can be transcribed and the transcript can be assembled into a pseudotyped retroviral vector particle.

22. A retroviral vector particle according to any one of embodiments 1 to 10 for use in a method of treatment of a human or animal subject.

23. A method of delivering a retroviral vector particle to a cell in vitro comprising mixing the cell with the retroviral vector particle of any one of embodiments 1 to 10.

24. A therapeutic vaccine comprising a retroviral vector particle according to any one of embodiments 1 to 10 and a pharmaceutically acceptable excipient.

Claims (30)

1. A lentiviral vector particle comprising:

(a) an envelope comprising the Sindbis virus E2 glycoprotein of SEQ ID NO.1, wherein 160X is absent or is an aliphatic amino acid selected from glycine, alanine, valine, leucine or isoleucine, or a variant of SEQ ID NO.1, said variant of SEQ ID NO.1 being amino acid residues 66-488 of SEQ ID NO.3, amino acid residues 66-488 of SEQ ID NO.4, or amino acid residues 66-486 of SEQ ID NO. 5; and

(b) a lentiviral vector genome comprising a sequence of interest.

2. The lentiviral vector particle of claim 1, wherein the E2 glycoprotein or variant binds to DC-SIGN.

3. The lentiviral vector particle of claim 1 or claim 2, wherein the 160X is absent or is glycine.

4. The lentiviral vector particle of claim 1 or claim 2, wherein 160X is glycine.

5. The lentiviral vector particle of claim 1 or claim 2, wherein the sequence of interest encodes a tumor-specific antigen or a virus-derived antigen.

6. The lentiviral vector particle of claim 1, wherein the lentiviral vector genome comprises a non-functional 3' LTR proximal polypurine channel.

7. The lentiviral vector particle of claim 5, wherein the tumor specific antigen is selected from the group consisting of: carbonic anhydrase IX, NY-ESO-1, MAGE, BAGE, RAGE, MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase related proteins, 5T4, SM22- α, carbonic anhydrase I, HIF-1 α, HIF-2 α, PSMA, PSA, STEAP, and NKX 3.1.

8. The lentiviral vector particle of claim 5, wherein the virus-derived antigen is an HIV antigen, an SIV antigen, an adenovirus antigen, an enterovirus antigen, a coronavirus antigen, a calicivirus antigen, a pestivirus antigen, an ebola virus antigen, a flavivirus antigen, a hepatitis virus antigen, a herpesvirus antigen, an infectious peritonitis virus antigen, an influenza virus antigen, a leukemia virus antigen, a marburg virus antigen, an orthomyxovirus antigen, a papilloma virus antigen, a parainfluenza virus antigen, a paramyxovirus antigen, a parvovirus antigen, a pestivirus antigen, a picornavirus antigen, a poliovirus antigen, a varicella virus antigen, a rabies virus antigen, a reovirus antigen, a retrovirus antigen, or a rotavirus antigen.

9. The lentiviral vector particle of claim 1, wherein the lentiviral vector genome comprises a nucleotide sequence encoding a dendritic cell maturation factor or a dendritic cell stimulatory factor, a non-functional 3' LTR proximal polypurine channel, and a U3 element lacking at least one of: enhancer sequences, TATA box, Sp1 site, NF-. kappa.B site, inactivated 3 'long terminal repeats, and self-inactivated 3' LTRs.

10. The lentiviral vector particle of claim 9, wherein the lentiviral vector genome comprises the nucleotide sequence of SEQ id No. 21, 22, or 23.

11. The lentiviral vector particle of claim 9, wherein the dendritic cell maturation factor or dendritic cell stimulatory factor is selected from the group consisting of: GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNF α, B7.1, B7.2, 4-1BB, CD40 ligand, and drug-inducible CD 40.

12. The lentiviral vector particle of claim 1 or claim 2, wherein expression of the sequence of interest is controlled by a group selected from the group consisting of: human ubiquitin-C promoter, cytomegalovirus early transient promoter, Rous sarcoma virus promoter, and tetracycline responsive promoter.

13. A lentiviral vector packaging system for making pseudotyped lentiviral vector particles, comprising:

(i) a first nucleic acid molecule encoding sindbis virus E2 glycoprotein of SEQ ID No.1, wherein 160X is absent or is an aliphatic amino acid selected from glycine, alanine, valine, leucine or isoleucine, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, said variant being amino acid residues 66-488 of SEQ ID No.3, amino acid residues 66-488 of SEQ ID No.4, or amino acid residues 66-486 of SEQ ID No. 5; and

(ii) a second nucleic acid molecule encoding gag and pol proteins;

(iii) a third nucleic acid molecule encoding a rev protein; and

(iv) a lentiviral vector genome comprising a sequence of interest.

14. The packaging system of claim 13, wherein the E2 glycoprotein has an amino acid sequence defined according to any one of claims 1, 3, and 4 above.

15. The packaging system of claim 13 or 14, wherein the pol protein has a non-functional integrase.

16. The packaging system of claim 15, wherein the non-functional integrase has the D64V mutation.

17. The packaging system of claim 14, wherein the lentiviral vector genome is a non-integrated lentiviral genome.

18. A method of making a packaging cell line comprising transfecting a host cell with the packaging system of any one of claims 14-17.

19. A method of using the packaging cell line of claim 18 to prepare lentiviral vector particles, comprising culturing the packaging cell line under conditions that allow formation of lentiviral vector particles.

20. A composition comprising lentiviral vector particles prepared according to the method of claim 19, comprising at least 10⁵IU/mL.

21. A method of making a lentiviral vector particle of any one of claims 1-12, comprising expressing in a cell

(i) A first nucleic acid molecule encoding sindbis virus E2 glycoprotein of SEQ ID No.1, wherein 160X is absent or is an aliphatic amino acid selected from glycine, alanine, valine, leucine or isoleucine, or a variant of sindbis virus E2 glycoprotein capable of infecting dendritic cells, said variant being amino acid residues 66-488 of SEQ ID No.3, amino acid residues 66-488 of SEQ ID No.4, or amino acid residues 66-486 of SEQ ID No. 5; and

(ii) a second nucleic acid molecule, wherein said second nucleic acid molecule can be transcribed and the transcript can be assembled into a pseudotyped lentiviral vector particle.

22. Use of a lentiviral vector particle according to any one of claims 1 to 12 in the preparation of a medicament for the treatment of a human or animal subject.

23. A method of delivering a lentiviral vector particle to a cell in vitro, comprising mixing the cell with the lentiviral vector particle of any one of claims 1-12.

24. A therapeutic or prophylactic vaccine comprising the lentiviral vector particles of any one of claims 1-12 and a pharmaceutically acceptable excipient.

25. A pharmaceutical composition comprising the lentiviral vector particle of any one of claims 1-12 and a pharmaceutically acceptable excipient.

26. Use of a lentiviral vector particle according to any one of claims 1-12 for the preparation of a medicament for inducing an immune response to an antigen in a subject.

27. An isolated nucleic acid encoding sindbis virus E2 glycoprotein of SEQ ID No.1, wherein 160X is absent or is an aliphatic amino acid selected from alanine, valine, leucine, or isoleucine, or a variant of SEQ ID No.1, said variant of SEQ ID No.1 being amino acid residues 66-488 of SEQ ID No.3, amino acid residues 66-488 of SEQ ID No.4, or amino acid residues 66-486 of SEQ ID No. 5.

28. The isolated nucleic acid of claim 27, wherein 160X is absent.

29. An expression vector comprising the nucleic acid of any one of claims 27-28.

30. A host cell comprising the expression vector of claim 29.