WO2006110344A1 - Novel methods for inducing an immune response against human immunodefiency virus - Google Patents

Abstract

The present invention relates to novel methods for inducing an immune response against human immunodeficiency virus. More particularly, the invention relates to the methods for inducing (or stimulating) both humoral and cellular immune response against HIV in a human subject.

Description

NOVEL METHODS FOR INDUCING AN IMMUNE RESPONSE AGAINST HUMAN IMMUNODEFIENCY VIRUS

FIELD OF THE INVENTION

The present invention generally relates to the fields of virology, microbiology, infectious disease and immunology. More particularly, the invention relates to novel methods for inducing an immune response against human immunodeficiency virus.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is a pathogenic retrovirus and the causative agent of acquired immune deficiency syndrome (AIDS) and related disorders (Barre-Sinossi et a/., Science 220:868-870 (1983); GaIIo et a/., Science 224:500-503 (1984)). Infection of human CD4⁺ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth and untimely death. Anti-viral therapeutic drugs that reduce viral burden and slow the progression to AIDS have become available, but are often prohibitively expensive for use in developing nations. Vaccines are an economically efficient means of controlling viral infections, and it is possible that a vaccine against HIV will be the most effective way of controlling the global AIDS crisis. Considerable progress has been made over the past several years in the development of an HIV vaccine, and a growing number of vaccine modalities are being investigated in pre-clinical and phase I/I I clinical trials. It is hypothesized that an efficacious HIV immunogenic composition should elicit both humoral and cellular immune responses. For example, cellular immune responses play an important role in controlling HIV replication, and humoral immune responses are necessary for controlling early viral replication and preventing viral entry into target cells (Calarota and Weiner, Expert Rev. Vaccines, 3(4), S135-149, 2004; Srivastava et a/., Expert Rev. Vaccines, 3(4), S33-52, 2004).

Recent protocols for stimulating both the humoral and cellular arms of the immune response against HIV have involved methods (or protocols) of administering an immune priming composition (i.e., a first immunogenic composition) and an immune boosting composition (i.e., a second immunogenic composition), wherein the priming and boosting compositions are heterologous compositions. For example, various anti-HIV immunogenic compositions are being tested in clinical trials using heterologous prime-boost immunization methodology (e.g., see Clinical Trials Section of the HIV Vaccine Trials Network (HVTN) web site accessible via the world wide web). For example, HVTN protocol number 049 comprises a priming immunization with a plasmid DNA (encoding gag and env) and a boosting immunization with a polypeptide (gp140), HVTN protocol number 057 comprises a priming immunization with plasmid DNA (encoding gag, pol, nef and env) and a boosting immunization with an adenovirus vector (VRC-H IVADVO 14-00-VP) and HVTN protocol number 026 comprises a priming immunization with attenuated canarypox virus (encoding env) and boosting with a polypeptide (gp120). However, it is often the case that immunogenic compositions that elicit certain types of HIV- specific immune responses may not elicit other important immune responses. Thus, there is currently a need for improved and/or novel methods for inducing both humoral and cellular immune responses against HIV in human subjects.

As of December 2004, the estimated number of people infected with human immunodeficiency virus (HIV) is more than 39 million, with 4.9 million people being newly infected in 2004 (Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO), "AIDS Epidemic Update", December 2004). More than eighty percent of the HIV infected individuals are living in developing nations such as sub-Saharan Africa (e.g., approximately 25.4 million infected) and Southeast Asia (e.g., approximately 7.1 million infected). Thus, there remains an urgent need to identify and develop effective preventative and therapeutic immunogenic compositions to curtail the global AIDS epidemic. A practical concern in developing countries is the lack of patient record-keeping. This may make it difficult to know whether a particular subject has previously received a priming composition or a boosting composition, or both. Therefore, for such subjects there is a need for a simplified immunization protocol which would reduce the need for detailed record keeping.

SUMMARY OF THE INVENTION The present invention broadly relates to novel methods for inducing an immune response against human immunodeficiency virus (HIV) in a human subject. More particularly, the invention relates to a method for inducing an immune response against HIV in a human subject comprising administering to the subject an immunogenic composition comprising the following components: (i) a DNA plasmid composition comprising a nucleotide sequence encoding an HIV polypeptide operably linked to a promoter and a polyadenylation signal and (ii) a polypeptide composition comprising one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof, wherein components (i) and (ii) of the immunogenic composition are co-administered at approximately the same time, wherein the immunogenic composition induces a humoral and a cellular immune response in the subject.

In certain embodiments, the method for inducing an immune response against HIV in a human further comprises one or more immune boosting administrations of the immunogenic composition, wherein components (i) and (ii) in each boosting administration are given at approximately the same time.

In certain embodiments, the DNA plasmid composition comprises a nucleotide sequence encoding an HIV polypeptide selected from the group consisting of Gag, Env, Nef, Vif, Tat, Pol, Rev, Vpr and Vpu. In one embodiment, the nucleotide sequence encodes a Gag polypeptide. In one particular embodiment, wherein the nucleotide sequence encoding the Gag polypeptide is derived from the HXB2 isolate of HIV. In still other embodiments, the nucleotide sequence encodes an Env polypeptide. In yet other embodiments, the nucleotide sequence encodes a Nef polypeptide.

In another embodiment, the DNA plasmid composition comprises a nucleotide sequence encoding at least two HIV polypeptides selected from the group consisting of Gag, Env, Nef, Vif, Tat, Pol, Rev, Vpr and Vpu. In one embodiment, the nucleotide sequence encoding the first HIV polypeptide and the nucleotide sequence encoding the second HIV polypeptides are comprised within the same DNA plasmid. In other embodiments, the nucleotide sequence encoding the first and the second HIV polypeptides are comprised within separate DNA plasmids. In certain embodiments, the nucleotide sequence encodes a Gag-Pol fusion polypeptide. In yet another particular embodiment, the nucleotide sequence encodes a Nef-Tat-Vif fusion polypeptide.

In certain embodiments, the promoter is selected from the group consisting of human cytomegalovirus (HCMV) immediate early promoter, the simian cytomegalovirus (SCMV) promoter, the murine cytomegalovirus (MCMV) promoter, the herpes simplex virus (HSV) latency-associated promoter-1 (LAP1), Simian virus 40 promoter, human elongation factor 1 alpha promoter and the human muscle cell specific desmin promoter. In another embodiment, the polyadenylation signal is selected from the group consisting of rabbit beta-globin poly(A) signal, synthetic polyA, HSV Thymidine kinase poly A, Human alpha globin poly A, SV40 poly A, human beta globin poly A, polyomavirus poly A and Bovine growth hormone poly A. In another embodiments, the polypeptide composition comprises one or more

HIV polypeptides selected from the group consisting of Env, Gag, Tat, Pol, Rev, Vpr and Vpu. In certain other embodiments, the polypeptide composition comprises one or more HIV polypeptide derived epitopes comprising an Env TH epitope, an Env CTL epitope, a Gag TH epitope, a Gag CTL epitope and a Nef CTL epitope. In one particular embodiment, the HIV polypeptide derived epitopes comprise one or more amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 8.

In still other embodiments, component (i) of the method further comprises a nucleotide sequence encoding an IL-12 polypeptide, wherein the IL-12 polypeptide is comprised of a p40 subunit and a p35 subunit. In one particular embodiment, the nucleotide sequence encoding the IL-12 polypeptide is a first nucleotide sequence encoding the IL-12 p40 subunit and a second nucleotide sequence encoding the IL- 12 p35 subunit, wherein the first and second nucleotide sequences are under the control of separate promoters. In another embodiment, the nucleotide sequence encoding the IL-12 polypeptide is comprised in a separate DNA plasmid than the DNA plasmid encoding the HIV polypeptide. In yet other embodiments, the nucleotide sequence encoding the IL-12 polypeptide is comprised in the same plasmid as the DNA plasmid encoding an HIV polypeptide. In other embodiments, component (i) of the method is formulated in a citrate buffer comprising bupivicaine, sodium chloride and a chelating agent, wherein the pH of the buffer is between 6.5 and 7.2.

In yet other embodiments, component (ii) of the method is formulated with an adjuvant. In one particular embodiment, the adjuvant is RC529-SE, GMCSF, or a combination thereof.

In another embodiment, components (i) and (ii) of the method are administered parenterally or mucosally. In one particular embodiment, the parenteral administration is by intramuscular injection. In another embodiment, components (i) and (ii) of the method are injected at the same intramuscular injection site. In still other embodiments, components (i) and (ii) of the method are co-formulated as a single injection dosage comprising both components (i) and (ii).

Other features and advantages of the invention will be apparent from the following detailed description, from embodiments thereof and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a schematic diagram of the plasmid DNA encoding RNA optimized HIV gag p37 polypeptide .

Figure 2 shows a schematic diagram of the dual promoter plasmid DNA encoding rhesus IL-12 p40 and rhesus IL-12 p35 subunits.

DETAILED DESCRIPTION OF THE INVENTION

The invention described hereinafter addresses a need in the art for methods of inducing immune responses against human immunodeficiency virus (HIV) in human subjects. More particularly, the invention described hereinafter has identified novel methods for inducing (or stimulating) both humoral and cellular immune responses against HIV in a human subject.

As detailed below in Example 2, the novel methods of the present invention provide a simplified immunization protocol for inducing a humoral and a cellular immune response against HIV in a human subject, wherein the methods comprise co-administering to the human subject an immunogenic composition comprising the following components: (i) a DNA plasmid composition comprising a nucleotide sequence encoding an HIV polypeptide operably linked to a promoter and a polyadenylation signal and (ii) a polypeptide composition comprising one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof, wherein components (i) and (ii) of the immunogenic composition are administered at approximately the same time. As demonstrated below in Example 2, immunization of macaques with HIV gag plasmid DNA was a potent inducer of cellular immune response, as measured by IFN-γ secretion, but not of humoral immune response, as measured by antigen- specific antibody production. In contrast, immunization of macaques with the multi- epitope TH-CTL polypeptides (i.e., SEQ ID Nos:1-4) was a potent inducer of humoral immune response, but not of cellular immune response. Surprisingly, co-immunizing macaques with both a gag plasmid DNA composition and the multi-epitope TH-CTL polypeptide composition (i.e., the polypeptides of SEQ ID Nos:1-4) resulted in robust cellular and humoral immune responses.

This result (i.e., inducing a humoral and cellular immune response) is particularly surprising, as it was previously believed in the art that co-immunizing a subject with both a plasmid DNA and a polypeptide component would result in unwanted competition between the two components, such that the immune response would be predominantly humoral or cellular, but not both. For example, it is generally accepted that the cytokine products of TM cells (e.g., IL-2, IFN-γ and TNF- α/β, which promote a cellular response) and the cytokine products of Th2 cells (e.g., 1L-4, 1L-5, IL-6, IL-10 and IL-13, which promote high antibody production) are mutually inhibitory for the differentiation and effector functions of the reciprocal phenotype (Mosmann and Sad, Immunology Today, (17): 138-146, 1996; Romagnani, Immunology Today, (18):263-266, 1997). Thus, the ability to co- administer an immunogenic composition comprising both a plasmid DNA and a polypeptide component, and concomitantly induce a humoral and cellular immune response, is considered highly advantageous.

Another advantage of co-administering the immunogenic composition of components (i) and (ii) as set forth in the present invention, in contrast to priming with component (i) and boosting with component (ii) or vice versa (as taught in the prior art), is the ability to immediately begin inducing both humoral and cellular immune responses, which may be particularly advantageous in the event that the subject does not return for one or more booster immunizations. A further advantage of co-administering the immunogenic composition of components (i) and (ii) as set forth in the present invention, in contrast to priming with component (i) and boosting with component (ii) or vice versa, is the elimination of the possibility that the subject will receive the priming composition in one or more booster immunizations, which could lead to an inadequate immune response in the subject.

A. PLASMlD DNA AND NUCLEIC ACID COMPONENTS

In certain embodiments the invention is directed to a method for inducing an immune response against HIV in a human subject comprising administering to the subject an immunogenic composition comprising a (i) DNA plasmid composition and a (ii) polypeptide composition, wherein components (i) and (ii) of the immunogenic composition are co-administered at approximately the same time, wherein the immunogenic composition induces a humoral and a cellular immune response in the subject. Thus, in particular embodiments, the invention is directed to a DNA plasmid component of the immunogenic composition. In certain embodiments, the DNA plasmid component of the immunogenic composition comprises a nucleotide sequence encoding an HIV polypeptide operably linked to a promoter and a polyadenylation signal. In particular embodiments, the DNA plasmid component comprises one or more nucleotide sequences encoding one or more HIV polypeptides, such as gag, pol, env, nef, vpr, vpu, vif and tat. In certain embodiments, nucleic acid sequences encoding the nef, tat and vif polypeptides are derived from the NL4-3 isolate of HIV and the nucleotide sequences encoding gag and pol are derived from the HXB2 isolate of HIV. The complete NL4-3 sequence is listed in the GenBank computer database under the Accession Number M19921. The complete HXB2 sequence is listed in the GenBank computer database under the Accession Number K03455. The nucleic acid sequence encoding the env polypeptide is listed in the GenBank computer database under the Accession Number AY612855 and bankit625244. One of skill in the art will appreciate that the sequence information is available in the art, and as such, can be used to clone genes for use in expressing polypeptides in DNA plasmids of the invention. Information on many sequences from HIV is available from the HIV sequence database at the Los Alamos National Laboratory and the National Center for Biotechnology Information at the United States National Library of Medicine, (8600 Rockville Pike, Bethesda, MD 20894).

In certain other embodiments, two or more genes from the HIV genome are cloned into a single expression plasmid, wherein gene fusions are prepared using full length gag-pol genes and nearly full length nef-tat-vif genes (U.S. Application No. 60/624,983, filed November 03, 2004, specifically incorporated herein by reference in its entirety). In one particular embodiment, the HIV genes used in the invention are RNA optimized (sequence modified) for enhanced polypeptide expression. See U.S. Patent Nos. 5,965,726; 5,972,596; 6,174,666; 6,291 ,664, 6,414,132 and 6,656,706. Alternatively, the HIV genes may be optimized in accordance with the methods provided in U.S. Application No. 60/576,819, filed on June 4, 2004. According to this method, the expression of genes is enhanced by replacing certain wild type codons with "surrogate" codons. The enhanced sequence of the polynucleotide is determined by selecting suitable surrogate codons. Surrogate codons are selected in order to alter the A and T (or A and U in the case of RNA) content in the wobble position in the codons of the naturally-occurring (wild-type) gene. The surrogate codons are those that encode the amino acids alanine, arginine, glutamic acid, glycine, isoleucine, leucine, proline, serine, threonine, and valine. Therefore, the modified nucleic acid sequence has surrogate codons for each of these amino acids throughout the sequence. For the remaining 11 amino acids, no alterations are made, thereby, leaving the corresponding naturally- occurring codons in place.

As defined hereinafter, the terms "plasmid", "plasmid DNA" and "DNA plasmid" are used interchangeably, and refer to the DNA or nucleic acid-based components of immunogenic compositions of the invention. As defined hereinafter, the term "plasmid" refers to a circular, supercoiled DNA molecule into which various nucleic acid molecules coding for regulatory sequences, open reading frames, cloning sites, stop codons, spacer regions or other sequences selected for structural or functional regions are assembled and used as a vector to express genes in a vertebrate host. Immunogenic compositions of this invention include a DNA plasmid comprising a DNA sequence encoding a selected antigen to which an immune response is desired. In the plasmid, the selected antigen is under the control of regulatory sequences directing expression thereof in a mammalian or vertebrate cell. The components of the plasmid itself are conventional.

Non-viral, plasmid vectors useful in this invention contain isolated and purified DNA sequences comprising DNA sequences that encode the selected immunogenic antigen. The DNA molecule may be derived from viral or non-viral, e.g., bacterial species that have been designed to encode an exogenous or heterologous nucleic acid sequence. Such plasmids or vectors can include sequences from viruses or phages. A variety of non-viral vectors are known in the art and may include, without limitation, plasmids, bacterial vectors, bacteriophage vectors, "naked" DNA and DNA condensed with cationic lipids or polymers.

Examples of bacterial vectors include, but are not limited to, sequences derived from bacille Calmette Guerin (BCG), Salmonella, Shigella, E. coli, and Listeria, among others. Suitable plasmid vectors include, for example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101 , pAC105, pVA51 , pKH47, pUB110, pMB9, pBR325, CoI E1 , pSC101 , pBR313, pML21 , RSF2124, pCR1 , RP4, pBAD18, and pBR328.

Examples of suitable inducible Escherichia coli expression vectors include pTrc (Amann et al., Gene, 69:301-315, 1988), the arabinose expression vectors (e.g., pBAD18, Guzman et al, J. Bacteriol., -/77:4121-4130, 1995), and pETIId (Studier et al., Methods in Enzymology, 185:60-89, 1990). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp- lac fusion promoter. Target gene expression from the pETIId vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase T7 gn1. This viral polymerase is supplied by host strains BL21 (DE3) or HMS I 74(DE3) from a resident prophage harboring a 17 gn1 gene under the transcriptional control of the lacUVδ promoter. The pBAD system relies on the inducible arabinose promoter that is regulated by the araC gene. The promoter is induced in the presence of arabinose

The promoter and other regulatory sequences that drive expression of the antigen in the desired mammalian host may similarly be selected from a wide list of promoters known to be useful for that purpose. A variety of such promoters are disclosed below. In an embodiment of the immunogenic DNA plasmid composition described below, useful promoters are the human cytomegalovirus (HCMV) promoter/enhancer (described in, e.g., U.S. Patent Nos. 5,158,062 and 5,385,839, incorporated herein by reference) and the SCMV promoter enhancer.

Additional regulatory sequences for inclusion in a nucleic acid sequence, molecule or vector of this invention include, without limitation, an enhancer sequence, a polyadenylation sequence, a splice donor sequence and a splice acceptor sequence, a site for transcription initiation and termination positioned at the beginning and end, respectively, of the polypeptide to be translated, a ribosome binding site for translation in the transcribed region, an epitope tag, a nuclear localization sequence, an IRES element, a Goldberg-Hogness "TATA" element, a restriction enzyme cleavage site, a selectable marker and the like. Enhancer sequences include, e.g., the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats or LTRs, etc. and are employed to increase transcriptional efficiency.

These other components useful in DNA plasmids, including, e.g., origins of replication, polyadenylation sequences (e.g., BGH polyA, SV40 polyA), drug resistance markers (e.g., kanamycin resistance), and the like may also be selected from among widely known sequences, including those described in the examples and mentioned specifically below.

Selection of promoters and other common vector elements are conventional and many such sequences are available with which to design the plasmids useful in this invention. See, e.g., Sambrook et a/, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1989) and references cited therein at, for example, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989). All components of the plasmids useful in this invention may be readily selected by one of skill in the art from among known materials in the art and available from the pharmaceutical industry. Selection of plasmid components and regulatory sequences are not considered a limitation on this invention.

Examples of suitable DNA plasmid constructs for use in immunogenic compositions are described in detail in the following patent publications, which are incorporated by reference herein for such disclosures, e.g., International Patent Publication Nos. WO99/43839 and WO98/17799; and United States Patent Nos. 5,593,972; 5,817,637; 5,830,876; and 5,891 ,505, among others. As set forth above, the DNA plasmid component comprises one or more nucleotide sequences encoding one or more HIV polypeptides, such as gag, pol, env, nef, vpr, vpu, vif and tat. In particular embodiments, the nucleic acid sequence encoding the one or more HIV polypeptides are optimized, such as by codon selection appropriate to the intended host (e.g., a human) and by removal of any inhibitory sequences, also discussed below with regard to antigen preparation.

In one embodiment, the plasmid component of the immunogenic composition is a plasmid encoding a single selected HIV antigen for expression in the host or one plasmid comprising a DNA sequence encoding more than one copy of the same selected antigen. Alternatively, the composition may contain one plasmid expressing multiple selected HIV antigens. Each HIV antigen may be under the control of separate regulatory elements or components. Alternatively, each HIV antigen may be under the control of the same regulatory elements. In still another embodiment, the DNA plasmid composition may contain multiple plasmids, wherein each DNA plasmid encodes the same or a different HIV antigen. See, for example, U.S. Application No. 60/624,983, filed November 03, 2004, specifically incorporated herein by reference in its entirety.

In a still further embodiment, the DNA plasmid immunogenic composition may further contain, as an individual DNA plasmid component or as part of the antigen-containing DNA. plasmid, a nucleotide sequence that encodes a desirable cytokine, lymphokine or other genetic adjuvant. Many such suitable adjuvants for which nucleic acid sequences are available, and are identified below in Section D.

Thus, in certain embodiments of the invention, component (i) of the immunogenic composition further comprises a nucleotide sequence encoding a human IL-12 polypeptide or a rhesus IL-12 polypeptide, wherein the IL-12 polypeptide is comprised of an IL-12 p40 subunit and an IL-12 p35 subunit (see, e.g., U.S. Patent Nos. 5,457,038; 5,648,467; 5,723,127 and 6,168,923, each incorporated by reference herein). In one embodiment exemplified in this invention, a plasmid DNA encoding the IL-12 polypeptide is a dual promoter plasmid (e.g., see FIG. 2) encoding the two rhesus IL-12 subunits. In another embodiment, a plasmid DNA encoding the IL-12 polypeptide is a dual promoter plasmid encoding the two human IL-12 subunits. For example, the Rhesus IL-12 plasmid is a dual promoter construct expressing the heterodimeric form of rhesus IL-12. The plasmid has a total of 6259 nucleotides. Each cistron in the plasmid contains one of the two interleukin 12 subunits, p35 or p40, under the control of separate regulatory elements. The p35 subunit is under the control of HCMV promoter/enhancer, and the SV40 polyadenylation signal (cloned between Sail and MIuI sites). The p40 subunit is under the control of the SCMV promoter and has a BGH polyadenylation signal (cloned into Xhol site).

The DNA plasmid compositions of the invention are desirably administered in a pharmaceutically acceptable diluent, excipient or carrier, such as those discussed below in Section D. Although the composition may be administered by any selected route of administration, in one embodiment a desirable method of administration is co-administration intramuscularly of an immunogenic composition comprising the one or more plasmids of the invention with bupivacaine as the facilitating agent and the one or more polypeptides and/or epitopes described below in Section B.

B. HIV POLYPEPTIDE ANTIGENS AND EPITOPES THEREOF

As described above, certain embodiments of the invention are directed to a method for inducing an immune response against HIV in a human subject comprising administering to the subject an immunogenic composition comprising a (i) DNA plasmid composition and a (ii) polypeptide composition, wherein components (i) and (ii) of the immunogenic composition are co-administered at approximately the same time, wherein the immunogenic composition induces a humoral and a cellular immune response in the subject. Thus, in particular embodiments, the invention is directed to a polypeptide component of the immunogenic composition. In certain embodiments, the polypeptide component of the immunogenic composition comprises one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof. In one particular embodiment, the polypeptide component of the immunogenic composition comprises one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof, wherein the HIV polypeptides are selected from gag, pol, env, nef, vpr, vpu, vif and tat. In certain embodiments, the one or more HIV polypeptides (or HIV polypeptide derived epitopes) are fusion polypeptides. In one particular embodiment, the fusion polypeptide is a Env-Gag fusion polypeptide. In another embodiment, the fusion polypeptide is a Env-Nef fusion polypeptide. The immunogenic composition comprising one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof may be administered with one or more adjuvants, such as those discussed below in Section D.

In one particular embodiment of the invention, the polypeptide component of the immunogenic composition comprises one or more TH-CTL polypeptides, also referred to as in the art as HLA-based (or HLA-derived) HIV immunogens or polypeptides, such as the HLA-based immunogenic compositions described in U.S. Patent No. 5,993,819; U.S. Application No. 10/753,339 (Publication No. US 2004/0197344) and International Application Publication No. WO 01/56355, each specifically incorporated herein by reference.

In one particular embodiment, the polypeptide component of the immunogenic composition comprises one or more T_H-CTL polypeptides set forth below in Table 1. In another embodiment, the polypeptide component of the immunogenic composition comprises all eight of the TH-CTL polypeptides listed in Table 1. It is contemplated herein, that an immunogenic composition comprising all eight of the T_H-CTL polypeptides listed in Table 1 , provides broad immunogenic coverage for the many different HLA allele types. In other embodiments, the polypeptide component of the immunogenic composition comprises TH-CTL polypeptides in addition to the eight T_H-CTL polypeptides listed in Table 1.

TABLE 1 TH-CTL PEPTIDE SEQUENCES

TH epitope - CTL epitope Peptide A: KQIINMWQEVGKAMYA - KAFSPEVIPMF (SEQ ID NO:1)

Peptide B: YKRWIILGLNKIVRMYS - NPPIPVGEIYKRWIILGLNKIVRMYSPTSI (SEQ ID NO:2)

Peptide C: DRVIEWQGAYRAIL - VGFPVRPQVPLRPMTYK (SEQ ID NO:3)

Peptide J: KQIINMWQWGKAMYA - GQMVHQAISPRTLNAWVKW (SEQ ID NO:4)

Peptide L: KQIINMWQWGKAMYA - EPFRDYVDRFYKTLRAEQASQEVKNWMTE (SEQ ID NO:5) Peptide M1 : KQIINMWQWGKAMYA -KIRLRPGGKKKYKLKHIVW (SEQ ID NO:6)

Peptide M2: KQIINMWQWGKAMYA - TGSEELRSLYNTVATLYCVHQKI (SEQ ID NO:7)

Peptide R: KQIINMWQWGKAMYA - SPAIFQSSMTKILEPFRKQNPDIVIYQYMDDL (SEQ ID NO:8) The TH-CTL polypeptides of the invention are designed based on available HLA databases and the description set forth in U.S. Application No. 10/753,339 (Publication No. US 2004/0197344) and International Application Publication No. WO 01/56355. Results obtained in International Histocompatibility Testing Workshops provide such a database (Histocompatibility Testing 1980, Teresaki (Ed.), UCLA Tissue Typing Laboratory, Los Angeles, Calif. (1980), Histocompatibility Testing 1984, Albert et al (Eds.), Springer-Verlag, Berlin (1984), lmmunobiology of HLA, 2 volumes, Dupont (Ed.), Springer-Verlag, New York, (1989), HLA 1991 , 2 volumes, Tsuji et al (Eds.), Oxford University Press, Oxford (1992)). The International Histocompatibility Workshop data (such as Histocompatibility Testing 1984, Albert et al (Eds.), Springer-Verlag, Berlin (1984), HLA 1991 , 2 volumes, Tsuji et al (Eds.), Oxford University Press, Oxford (1992)), supplemented with published data from selected laboratories provide an estimate of the frequencies of HLA alleles that have been shown to serve as restriction elements for HIV CTL epitopes (Williams et al, Human Immunol. 33:39-46 (1992), Chandanayingyong et al, In Proceedings of the Second Asia and Oceania Histocompatibility Workshop Conference, Simons et al (Eds.), Immunopublishing, Toorak, pgs. 276-287 (1983)) HIV Molecular Immunology Database (1995), Korber et al (Eds.), Los Alamos National Laboratory: Published by Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. 87545).

U.S. Patent No. 5,993,819 includes a description of the steps involved in the development of an HLA-based HIV immunogenic composition and presents the following general formula: Tm-X-i, T_H2-X2_> T_H3-X3, . . . Tn_n-X_n. where TH = immunodominant T helper epitopes and X = MHC Class I CTL epitopes. For example, the strategy that can be used in this analysis is to first identify the most frequent restriction elements in the population under consideration for immunization (or common to the 4 populations), to identify peptides that are presented by more than one HLA allele, and then to seek commonality between these two lists. Probability calculations then utilize the frequencies of the commonality alleles supplemented by those of additional high frequency alleles in the population. Alleles can be added until the proportion of the individuals in the population carrying one or more of the alleles in the list is at an acceptable level, for instance, greater than 90% in the examples. The aim is to maximize the sum of the HLA gene frequencies that recognize the least number of different HIV peptides to be included in an HIV immunogen. The next step is to choose the peptides associated with the restricting allele. In some instances, only one peptide is associated with an allele while in others, multiple peptides are presented by the same allele.

C. DOSAGES, ROUTES AND TIMING OF ADMINISTRATION FOR IMMUNOGENIC

COMPOSITIONS

As described above, the invention relates to a method for inducing an immune response against HIV in a human subject, particularly a humoral and a cellular immune response in the human subject. The method of the invention comprises "co-administering" to the human subject an immunogenic composition comprising the following components: (i) a DNA plasmid composition comprising a nucleotide sequence encoding an HIV polypeptide operably linked to a promoter and a polyadenylation signal and (ii) a polypeptide composition comprising one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof.

As defined herein, the terms "co-administer", "co-immunize" and "co-deliver" are used interchangeably, and refer to the process of administering to the subject, "at the same time" or as approximately close to the same time as possible, an immunogenic composition of components (i) and (ii), as set forth above.

The immunogenic composition comprising components (i) and (ii) may be formulated together (i.e., co-formulated) and administered to the subject as a single dose (e.g., a single syringe dosage for injection). When co-formulated as a single dose, co-administration of immunogenic composition of components (i) and (ii) to the human subject is concomitantly "at the same time".

Alternatively, the immunogenic composition comprising components (i) and

(ii) may be formulated separately (e.g., component (i) formulated in one syringe and component (ii) formulated in a second syringe). In these embodiments, components

(i) and (ii), which are separately formulated, are to be co-administered "at approximately the same time".

The immunogenic compositions of this invention are administered to a human by a variety of routes including, but not limited to, intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal (see, e.g., International patent publication No. WO 98/20734, which is hereby incorporated by reference), intramuscular, intraperitoneal, subcutaneous, intravenous and intraarterial. The appropriate route is selected depending on the nature of the immunogenic composition used, and an evaluation of the age, weight, sex and general health of the patient and the antigens present in the immunogenic composition, and similar factors by an attending physician or other qualified health professional.

In certain embodiments of the invention, components (i) and (ii) are administered parenterally or mucosally. In one particular embodiment, components (i) and (ii) are administered by intramuscular injection. In another embodiment, when components (i) and (ii) are separately formulated, they are injected at the same intramuscular injection site.

In certain embodiments, the method for inducing an immune response against HIV in a human further comprises one or more immune boosting administrations of the immunogenic composition, wherein components (i) and (ii) in each boosting administration are given at approximately the same time.

In general, selection of the appropriate "effective amount" or dosage for the components of the immunogenic composition(s) of the present invention will also be based upon the identity of the selected antigens in the immunogenic composition(s) employed, as well as the physical condition of the subject, most especially including the general health, age and weight of the immunized subject. The method and routes of administration and the presence of additional components in the immunogenic compositions may also affect the dosages and amounts of the DNA plasmid compositions. Such selection and upward or downward adjustment of the effective dose is within the skill of the art. The amount of plasmid and/or polypeptide required to induce an efficacious immune response in the patient without significant adverse side effects varies depending upon these factors. Suitable doses are readily determined by persons skilled in the art.

D. CARRIERS, DILUENTS, FACILITATING AGENTS, ADJUVANTS AND FORMULATIONS USEFUL FOR THE IMMUNOGENIC COMPOSITIONS OF THIS INVENTION

The immunogenic compositions of the invention further comprise a pharmaceutically acceptable diluent, excipient or a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable diluent is sterile water, sterile isotonic saline or a biological buffer. The antigenic compositions may also be mixed with such diluents or carriers in a conventional manner. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. The appropriate carrier is evident to those skilled in the art and will depend in large part upon the route of administration.

In certain embodiments of the invention, component (i) of the invention is formulated in a buffer having a pKa of about 6.0 to about 7.5. In one particular embodiment, component (i) is formulated in a citrate buffer having a pH between 6.5 and 7.2. In certain other embodiments, the citrate buffer further comprises sodium chloride, a chelating agent, bupivicaine, or a combination thereof. In certain other embodiments of the invention, component (ii) is formulated with the adjuvant RC529- SE, GMCSF or a combination thereof. Still additional excipients that may be present in the immunogenic compositions of this invention are adjuvants, transfection facilitating agents, preservatives, surface active agents, and chemical stabilizers, suspending or dispersing agents. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the human or veterinary subjects.

1. ADJUVANTS

An adjuvant is a substance that enhances the immune response when administered together with an immunogen or antigen. A number of cytokines or lymphokines have been shown to have immune modulating activity, and thus may be used as adjuvants, including, but not limited to, the interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the interferons-α, β and Y, granulocyte-macrophage colony stimulating factor (GMCSF, see, e.g., U.S. Patent No. 5,078,996 and ATCC Accession Number 39900), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), and the tumor necrosis factors α and β (TNF). Still other adjuvants useful in this invention include a chemokine, including without limitation, MCP-1 , MIP-1α, MIP-1β, and RANTES. Adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin and E-selectin may also be useful as adjuvants.

Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1 , a member of the integrin family such as LFA-1 , VLA-1 , Mac-1 and p150.95, a member of the immunoglobulin superfamily such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3, co- stimulatory molecules such as CD40, CD40L, B7.1 and B7.2, growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 , and vascular endothelial growth factor, receptor molecules including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6.

Still another adjuvant molecule includes Caspase (ICE). See, also International Patent Publication Nos. WO98/17799 and WO99/43839, incorporated herein by reference.

Suitable adjuvants used to enhance an immune response include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, which is hereby incorporated by reference. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6,113,918, which is hereby incorporated by reference. One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-O- phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoyl-aminoJ-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). This 529 adjuvant is formulated as an aqueous form or as a stable emulsion (RC529-SE).

Still other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene, D-lactide-polylactide/glycoside, pluronic polyols, muramyl dipeptide, killed Bordetella, saponins, such as Stimulon™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No. 5,057,540, which is hereby incorporated by reference, and particles generated therefrom such as ISCOMS (immunostimulating complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646, which is hereby incorporated by reference), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, incorporated herein by reference.

Also useful as adjuvants are cholera toxins and mutants thereof, including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid (other than aspartic acid), preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36). In some embodiments, plasmid DNA that encodes an adjuvant may be administered in an immunogenic composition. In such cases, an adjuvant whose DNA is inserted into a plasmid for inclusion in the immunogenic compositions of the invention includes, but are not limited to, interleukin-1 (IL-1), IL-5, IL-10, IL-12, IL-15, IL-18, TNF-α, TNF-β and BL-1 (as described in published International Patent Application WO 98/17799); B7.2 (as described in published International Patent Application WO 00/51432); IL-8, RANTES, G-CSF, IL-4, mutant IL-18, IL-7, TNF-R (as described in published International Patent Application WO 99/43839); and mutant CD80 (as described in published International Patent Application WO 00/66162). As used herein, the term "IL-12 protein" is meant to refer to one or both human IL-12 subunits including single chain IL-12 proteins in which the two subunits are encoded by a single coding sequence and expressed as a single protein having a linker sequences connecting the two subunits. In one embodiment, the desired adjuvant is IL-12 protein, which is expressed from one plasmid (both subunits) or from two plasmids (one subunit per plasmid). See, e.g., US Patent Nos. 5,457,038; 5,648,467; 5,723,127 and 6,168,923, incorporated by reference herein. In one embodiment, the cytokine may be administered as a protein. As used herein, the term "IL-12 protein" is meant to refer to one or both IL-12 subunits, as well as single chain IL-12 proteins in which the two subunits are encoded by a single coding sequence and expressed as a single protein having a linker sequence connecting the two subunits. In another embodiment, a plasmid encoding and expressing IL-15 is administered instead of a plasmid encoding and expressing IL-12.

In a particular embodiment, the cytokine is administered as a nucleic acid composition comprising a DNA sequence encoding the cytokine under the control of regulatory sequences directing expression thereof in a mammalian cell. In still another embodiment, the cytokine-expressing plasmid is administered with the DNA plasmid encoding selected antigens in an immunogenic composition. In still another embodiment, the cytokine is administered between the administrations of a priming immunogenic composition and a boosting immunogenic composition. In yet another embodiment, the cytokine is administered with the boosting step. In still another embodiment, the cytokine is administered with both priming and boosting compositions.

In certain embodiments of the invention, CpG DNA may be included in the plasmid as an adjuvant. As used herein, CpG DNA refers to an oligonucleotide containing at least one unmethylated CpG dinucleotide nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e. "CpG DNA") or DNA containing a 5' cytosine followed by 3' guanosine and linked by a phosphate bond) and activates the immune system. See U.S. Patent 6,406,705 to Davis et a/., and U.S. Patent No. 6,207,646 to Krieg et a/., which are hereby incorporated by reference in their entirety. CpG DNA from bacterial DNA, but not vertebrate DNA₁ has direct immunostimulatory effects on peripheral blood mononuclear cells (PBMC) in vitro. This lymphocyte activation is due to unmethylated CpG dinucleotides, which are present at the expected frequency in bacterial DNA (1/16), but are under- represented (CpG suppression, 1/50 to 1/60) and methylated in vertebrate DNA. It is has been suggested that the rapid immune activation in response to CpG DNA may have evolved as one component of the innate immune defense mechanisms that recognize structural patterns specific to microbial molecules. See U.S. Patent 6,406,705 to Davis et a/., and U.S. Patent No. 6,207,646 to Krieg et a/., which are hereby incorporated by reference in their entirety.

2. FACILITATING AGENTS OR CO-AGENTS

Immunogenic compositions composed of polynucleotide molecules often contain optional excipients such as polynucleotide transfection facilitating agents or "co-agents", such as a local anesthetic, a peptide, a lipid including cationic lipids, a liposome or lipidic particle, a polycation such as polylysine, a branched, three- dimensional polycation such as a dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or another compound that facilitates polynucleotide transfer to cells. Such a facilitating agent includes the local anesthetic bupivacaine or tetracaine (see U.S. Patent Nos. 5,593,972; 5,817,637; 5,380,876; 5,981 ,505 and 6,383,512 and International Patent Publication No. WO98/17799, which are hereby incorporated by reference). Other non-exclusive examples of such facilitating agents or co-agents useful in this invention are described in U. S. Patent Nos. 5,703,055; 5,739,118; 5,837,533; International Patent Publication No. WO96/10038, published April 4, 1996; and International Patent Publication No WO94/16737, published August 8, 1994, which are each incorporated herein by reference.

Typically, the transfection facilitating agent is present in an amount that forms one or more complexes with the nucleic acid molecules. When the transfection facilitating agent is mixed with nucleic acid molecules or plasmids of this invention, it forms a variety of small complexes or particles that pack the DNA and are homogeneous. Thus, in one embodiment of the immunogenic compositions of this invention, the complexes are formed by mixing the transfection facilitating agent and at least one plasmid of this invention.

In a particular embodiment, an immunogenic composition of the invention may be comprised of more than one type of plasmid. Alternatively, in another embodiment of the compositions of the invention, the transfection facilitating agent may be pre-mixed with each plasmid separately. The separate mixtures are then combined in a single composition to ensure the desired ratio of the plasmids is present in a single immunogenic composition, if all plasmids are to be administered in a single bolus administration. Alternatively, the transfection facilitating agent and each plasmid may be mixed separately and administered separately to obtain the desired ratio.

3. OTHER ADDITIVES TO THE IMMUNOGENIC COMPOSITIONS Other excipients can be included in the immunogenic compositions of this invention, including preservatives, stabilizing ingredients, surface active agents, and the like. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.

Suitable stabilizing ingredients that may be used include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.

Suitable surface active substances include, without limitation, Freunds incomplete adjuvant, quinone analogs, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide), methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl peptide and dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum phosphate, etc. and immune stimulating complexes (ISCOMS). The plasmids may also be incorporated into liposomes for use as an immunogenic composition. The immunogenic compositions may also contain other additives suitable for the selected mode of administration of the immunogenic composition. The immunogenic composition of the invention may also involve lyophilized polynucleotides, which can be used with other pharmaceutically acceptable excipients for developing powder, liquid or suspension dosage forms. See, e.g., Remington: The Science and Practice of Pharmacy, Vol. 2, 19^th edition (1995), e.g., Chapter 95 Aerosols; and International Patent Publication No. WO99/45966, the teachings of which are hereby incorporated by reference.

These immunogenic compositions can contain additives suitable for administration via any conventional route of administration. In some embodiments, the immunogenic composition of the invention is prepared for administration to human subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Thus, the immunogenic compositions may also include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations. In one embodiment of the invention, the immunogenic compositions are prepared as a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Other useful parenterally- administrable formulations include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

The immunogenic compositions of the present invention, are not limited by the selection of the conventional, physiologically acceptable carriers, diluents and excipients such as solvents, buffers, adjuvants, facilitating agents or other ingredients useful in pharmaceutical preparations of the types described above. The preparation of these pharmaceutically acceptable compositions, from the above- described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art.

E. EXAMPLES

The following examples are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The following examples are presented for illustrative purpose, and should not be construed in any way limiting the scope of this invention. EXAMPLE 1 MATERIALS AND METHODS

Plasmids

A schematic diagram of the plasmid DNA expressing HIV-1 Gag polypeptide is set forth in FIG. 1. The nucleic sequence encoding the gag polypeptide was derived from the HXB2 isolate of HIV-1. The HIV gag plasmid encodes a C- terminally truncated HIV HXB2 gag gene (p37) under control of the human cytomegalovirus (HCMV) immediate early promoter and bovine growth hormone (BGH) polyadenylation signal. P37 corresponds to HIV gag p17 and p24 regions of HIV Gag protein. The HIV gag p37 gene was RNA optimized by introducing multiple point mutations within the coding region inactivating endogenous inhibitory sequences thus allowing for high level Rev independent expression of the HIV Gag p37 protein. HIV Gag p37 expression by the plasmid was confirmed by Western blot after transient transfection of human rhabdosarcoma (RD) cells (data not shown). A schematic diagram of the plasmid DNA expressing IL-12 is set forth in FIG.

2, which is a dual promoter IL-12 expression vector. The dual promoter IL-12 expression vector encodes the rhesus IL-12 p35 and p40 genes. (Rhesus IL-12 p35 and p40 gene fragments used for cloning were kindly provided by Dr. F. Villinger, Emory University School of Medicine, Atlanta GA. The rhesus IL-12 p35 subunit was expressed under control of the HCMV immediate early promoter and SV40 polyadenylation signal, while the rhesus IL-12 p40 subunit was expressed under control of the simian CMV promoter (SCMV) and BGH polyadenylation signal. Production of rhesus IL-12 was confirmed after transient transfection of RD cells by screening cell supematants using an anti-human IL-12 p70 capture ELISA (data not shown; Endogen, Woburn, MA). Bioactivity of the plasmid-expressed rhesus IL-12 was confirmed by assaying supematants from transiently transfected RD cells for their capacity to induce IFN-γ secretion in resting rhesus peripheral blood lymphocytes (PBLs; data not shown).

The HIV-1 gag and IL-12 plasmids were manufactured by Puresyn Inc. (Malvern, PA). Plasmids were propagated in E. coli, isolated from cells by alkaline lysis, purified by column chromatography and formulated individually at a concentration of 2.5 mg/mL in isotonic citrate buffer (29.3 mM sodium citrate, 0.67 mM citric acid, 15OmM NaCI, 0.34 mM EDTA, pH 6.4-6.7) containing 0.25% bupivacaine to allow for the formation of DNA:bupivacaine complexes. Final plasmid preparations consisted of greater than 90% supercoiled plasmid DNA and residual endotoxin was shown to be less than 30 EU/mg DNA (data not shown).

TH-CTL Polypeptides

The TH-CTL polypeptide composition comprised four HIV-1 derived polypeptides set forth as SEQ ID NO:1 through SEQ ID NO:4 (e.g., see FIG. 3). The polypeptides were selected based on prediction of recognition by HLA alleles (e.g., see U.S. Patent Application No. 10/753,339, (Publication No. US 2004/0197344) and International Publication No. WO 01/56,355), each specifically incorporated herein by reference). Each polypeptide consists of a T helper (T_H) sequence and a selected CTL epitope. The CTL epitopes include sequences identified from HIV Gag, Nef, and Env proteins. The TH-CTL polypeptide composition (i.e., comprising all four T_H-CTL polypeptides of SEQ ID NO: 1-4) was formulated and administered at a final volume of 1 mL, comprising 1 mg of total polypeptide, 50 μg RC-529SE (Corixa; Hamilton, MT) and 250 μg GM-CSF (commercial name Leukine™, manufactured by Berlex Laboratories; Richmond, CA).

Animals A total of eighteen genetically unselected cynolmogus macaques were used in the immunogenicity studies set forth below in Example 2. The macaqges were housed at New Iberia Research Center (New Iberia, LA) and maintained in accordance with the Guide for the Care and Use of Laboratory animals (National Research Council, National Academic Press, Washington, DC, 1996).

Immunization Regimen

Set forth below in Table 2 are the prime-boost immunization regimens tested in the present invention. Regimen number 1 comprised (a) a priming immunization with the TH-CTL polypeptide composition and (b) a boosting immunization with the gag plasmid DNA composition. Regimen number 2 comprised (a) a priming immunization with the gag plasmid DNA composition and (b) a boosting immunization with the T_H-CTL polypeptide composition. Regimen 3 comprised (a) a priming co-immunization with the gag plasmid DNA composition and the TH-CTL polypeptide composition and (b) a boosting co-immunization with the gag plasmid DNA composition and T_H-CTL polypeptide composition. Regimen 4 was a control in which the macaques were not immunized.

It should be noted in Regimen 1-3, that whenever the gag plasmid DNA was administered, the composition was co-formulated with the rhesus dual promoter plasmid expressing both IL-12 subunits (e.g., see FIG. 2). Thus, throughout Examples 1 and 2, the use of terms gag plasmid, HIV gag and gag plasmid DNA include in their formulation, the dual promoter plasmid expressing IL-12. The HIV gag and IL-12 plasmids were therefore pre-mixed and given as 1.2 mL immunization into the macaque quadriceps muscles via needle and syringe inoculation. For the co-immunization regimen (i.e., Regimen No. 4), the gag plasmid DNA composition and the TH-CTL polypeptide composition were formulated separately and a half-dose of each injected into both quadriceps (i.e., (a) Y≥ dose gag plasmid + Y∑ dose TH-CTL into one quadricep and (b) Vz dose gag plasmid + 34 dose TH-CTL into the other quadricep).

TABLE 2 IMMUNIZATION REGIMEN

IFN-γ ELISPOT Assay

Ninety-six-well flat-bottomed plates were coated with an anti-gamma interferon (IFN-γ) monoclonal antibody (clone B27, Pharmingen), washed, and blocked with 5% fetal bovine serum in PBS for 2 hours at 37⁰C. Peripheral blood lymphocytes (PBLs) were resuspended in complete culture media (5%FBS in RPMI) containing either 100 μg/mL PHA-M, 2 pools of 15-mer overlapping by 11 amino acid peptides comprising the HXB2 HIV gag polypeptide sequence, the T_H-CTL polypeptide composition of SEQ ID NO: 1-4 at 2.5 μM, or media alone. Input cell numbers were 2.0 * 10⁵ PBLs in 100 μL/well in duplicate wells. Cells were then incubated for 18-20 hours at 37°C, after which time the cells were removed from the plate. Anti-human IFN-γ antibody (Biosource; Catalogue No. N0N0105) conjugated with Biotin was added to the wells at 6.5 μg/mL, 100 μL/well for 2 hours at 37°C, followed by Strepavidin-HRP (Becton Dickinson, Catalogue No. 51-900209) for one hour at room temperature. The plates were then developed with AEC kit (Becton

Dickinson, Catalogue No. 55-1951). The plates were then washed and dried, and spots were enumerated with the CTL software (CTL; Cleveland Ohio).

Antibody Titers bv ELISA

ELISA plates were coated with HIV Gag p24 protein (20 ng/well) or T_H-CTL polypeptide (20 ng/well). Serum samples were added to wells at a starting dilution of 1 :64 in 1%BSA/1xPBS, and diluted 2-fold across the ELISA plates. A Biotin conjugated primary antibody specific for monkey IgG diluted in 1%BSA/1xPBS at 1 :30,000 (Accurate Scientific; Westbury NY), followed by 1 :10,000 SA-HRP (Roche; Catalogue No. 1089153) diluted in 1%BSA/1xPBS was added to the plates. Plates were developed with TMB (Sigma) and OD was read at 450nm. The reported antigen-specific antibody titer is the reciprocal of the last dilution giving a higher reading than the same animal's naϊve serum titer + 3 standard deviations.

CBC Panel

Complete blood counts (CBC) was determined using a STKS Hematology analyzer at the UL Lafayette-NIRC Clinical Pathology Laboratory according to New

Iberia Research Center SOP NO.: L-HOI.04. The normal range for each parameter, as determined by New Iberia Research Center (NIRC) internal standard is as follows:

Hematocrit, 35.9-41.9%; White cell count, 5.8-13.8 x 10³/mm³; Platelet, 311-511 x

10³/mm³; Lymphocytes, 18.8-54.8 %; Red blood cell, 4.72-5.92 x10⁶/mm³; Hemoglobin, 11.0-14.0 gm/dL. Statistical analysis

ANOVA analysis of IFN-γ ELISpot data was performed on SAS version 8.2 software, using type 1 GENMOD procedure with negative binomial distribution. Log10 transformed ELISA was analyzed using the SAS version 8.2 software, the least square means of each group were compared using the Mixed procedure.

EXAMPLE 2

CELLULAR AND HUMORAL IMMUNE RESPONSES TO PRIME-BOOST IMMUNIZATIONS

CELLULAR IMMUNE RESPONSE TO IMMUNIZATION

The cellular immune response to a particular prime-boost immunization regimen set forth above in Table 2 was measured using the ELISpot assay to quantitate the number of IFN-γ secreting cells responding to the immunogens. The results demonstrated (Table 3) that following three priming immunizations (3x) with either gag plasmid DNA alone (Regimen 2), or three priming co-immunizations (3x) with the gag plasmid DNA + the T_N-CTL polypeptide composition (Regimen 3), the macaques were able to respond to antigen stimulation by secretion of IFN-γ. Additionally, both regimen 2 and regimen 3 were able to further boost IFN-γ SFU when the three booster immunizations were administered (Table 3).

TABLE 3 TOTAL ANTIGEN-SPECIFIC IFN-/ ELISPOT (SPOT FORMING CELLS/10⁶ PBMC)

TABLE 3 (CONTINUED) TOTAL ANTIGEN-SPECIFIC IFN-γ ELISPOT (SPOT FORMING CELLS/10⁶ PBMC)

PBMC from macaques were re-stimulated with 2.5mM of 15mer-overlapping by 11 peptide pools. The IFN-γ spot-forming units (SFU)/10⁶ PBMC (y-axis) were enumerated at different time points during the study. The three priming immunizations were given at 0 weeks, 4 weeks and 8 weeks. The three boosting immunizations were given at week 20, week 24 and week 28

However, the priming and boosting immunization effect of the TH-CTL polypeptide immunization to all tested antigens was somewhat weak, as demonstrated by the reduced IFN-γ SFU compared to gag plasmid DNA, or gag plasmid DNA + T_H-CTL polypeptide immunization at peak post-priming (Table 4) and post-boosting time points (Table 5).

TABLE 4 STATISTICAL COMPARISON OF POST-PRIME IFN^y ELISPOT RESPONSES

Comparison of IFN-γ SFU between different immunization groups at week 10, peak post-prime.

TABLE 5 STATISTICAL COMPARISON OF POST-BOOST IFN-γ ELISPOT RESPONSES

Comparison of IFN-γ SFU between different immunization groups at week 30, peak post-boost.

When macaques primed with the TR-CTL polypeptide composition and boosted with gag plasmid DNA were compared to macaques that were only primed with gag plasmid DNA, it was observed that the T_H-CTL-primed, gag plasmid DNA- boosted group displayed higher responder rates (4/4 to 2/4; Table 6)) and higher ELISpot SFU (1771 SFU/10⁶PBMC to 683 SFU/10⁶PBMC; Table 6). Although the difference did not achieve statistical significance, this result indicated that T_H-CTL polypeptide priming was able to confer immunological memory that enabled enhanced immune response upon subsequent gag plasmid DNA boosting.

TABLE 6

COMPARISON BETWEEN PLASMID IMMUNIZED NAΪVE GROUP AND 3X PEPTIDE PRIMED 3X

PLASMID BOOSTED GROUP (P=O-138)

HUMORAL IMMUNE RESPONSE TO IMMUNIZATION

ELlSA assays were performed to assess the induction of IgG antibodies specific for the administered antigens. The results indicated that animals receiving the TH-CTL polypeptide composition (formulated with RC529 and GM-CSF), delivered alone, or in combination with gag plasmid DNA, were able to achieve higher endpoint titers to both HIV gag (Table 7) and TH-CTL (Table 8) polypeptide antigens, compared to animals given gag plasmid DNA, either as priming or boost.

TABLE 7 TOTAL HIV P24 GAG-SPECIFIC ELISA (LOG10)

TABLE 7 (CONTINUED)

ELISA results for HIV Gag p24-specific endpoint titers for all immunization groups are represented The three priming immunizations were given at 0 weeks, 4 weeks and 8 weeks. The three boosting immunizations were given at week 20, week 24 and week 28.

TABLE 8 TOTAL T_N-CTL PEPTIDE-SPECIFIC ELISA (LOG10)

ELISA results for HIV CTL peptide-specific endpoint titers for all immunization groups are represented The three priming immunizations were given at 0 weeks, 4 weeks and 8 weeks. The three boosting immunizations were given at week 20, week 24 and week 28.

A statistical comparison of peak post-prime endpoint titers at week 10 is shown in Tables 9 and 10, which demonstrates that the T_H-CTL polypeptide composition induces a more robust humoral immune response which is superior to the plasmid composition in eliciting antibodies specific for the immunization antigen.

TABLE 9 STATISTICAL COMPARISON OF POST-PRIME (W10) P24 HlV GAG ELISA RESPONSES

GMT(LOGiO GEOMEAN TITER)

TABLE 10 STATISTICAL COMPARISON OF POST-PRIME (w10) T_N-CTL PEPTIDE ELISA RESPONSES

GMT(LOGiO GEOMEAN TITER) At peak post-boost response (week 30), a similar trend is observed with the immunization groups containing the T_H-CTL polypeptide composition possessing the higher endpoint tiers, Tables 11 and 12).

TABLE 11 STATISTICAL COMPARISON OF POST-BOOST (W30) P24 HIV GAG ELISA RESPONSES

GMT(LOGiO GEOMEAN TITER)

TABLE 12 STATISTICAL COMPARISON OF POST-BOOST (W30) T_H-CTL PEPTIDE ELISA RESPONSES

GMT(LOGiO GEOMEAN TITER)

CBC PROFILE OF MACAQUES

A complete blood count (CBC) analysis was performed on peripheral blood obtained from each macaque over the course of the immunization regimen. The results obtained showed that the plasmid cytokine adjuvants were well tolerated as all parameters tested were within the acceptable range established by New Iberia

Research Center (data not shown). In addition, immunization of rhesus macaques with plasmids formulated with bupivicaine did not adversely affect CBC parameters (data not shown). Additionally, no adverse events, local injection site reactivity, loss of appetite, general appearance or behavior related to the administration of the immunogenic composition was reported during the course of the study.

Claims

What is Claimed is:

1. A method for inducing an immune response against human immunodeficiency virus (HIV) in a human subject, the method comprising administering to the subject an immunogenic composition comprising the following components:

(i) a DNA plasmid composition comprising a nucleotide sequence encoding an HIV polypeptide operably linked to a promoter and a polyadenylation signal, and (ii) a polypeptide composition comprising one or more HIV polypeptides, one or more HIV polypeptide derived epitopes, or a combination thereof, wherein components (i) and (ii) of the immunogenic composition are co-administered at approximately the same time, wherein the immunogenic composition induces a humoral and a cellular immune response in the subject.

2. The method of claim 1, further comprising one or more immune boosting administrations of the immunogenic composition, wherein components (i) and (ii) in each boosting administration are given at approximately the same time.

3. The method of claim 1 , wherein the DNA plasmid composition comprises a nucleotide sequence encoding an HIV polypeptide selected from the group consisting of Gag, Env, Nef, Vif, Tat, Pol, Rev, Vpr and Vpu.

4. The method of claim 3, wherein the nucleotide sequence encodes a Gag polypeptide.

5. The method of claim 4, wherein the nucleotide sequence encoding the Gag polypeptide is derived from the HXB2 isolate of HIV.

6 The method of claim 3, wherein the nucleotide sequence encodes an Env polypeptide.

7. The method of claim 3, wherein the nucleotide sequence encodes a Nef polypeptide.

8. The method of claim 1 , wherein the DNA plasmid composition comprises a nucleotide sequence encoding at least two HIV polypeptides selected from the group consisting of Gag, Env, Nef, Vif, Tat, Pol, Rev, Vpr and Vpu.

9. The method of claim 8, wherein the nucleotide sequence encoding the first HIV polypeptide and the nucleotide sequence encoding the second HIV polypeptides are comprised within the same DNA plasmid.

10. The method of claim 8, wherein the nucleotide sequence encoding the first and the second HIV polypeptides are comprised within separate DNA plasmids.

11. The method of claim 8, wherein the nucleotide sequence encodes a Gag-Pol fusion polypeptide.

12. The method of claim 8, wherein the nucleotide sequence encodes a Nef-Tat- Vif fusion polypeptide.

13. The method of claim 12, wherein the nucleotide sequence encoding the Nef- Tat-Vif fusion polypeptide is derived from the NL4-3 isolate of HIV.

14. The method of claim 1 , wherein the promoter is selected from the group consisting of human cytomegalovirus (HCMV) immediate early promoter, the simian cytomegalovirus (SCMV) promoter, the murine cytomegalovirus (MCMV) promoter, the herpes simplex virus (HSV) latency-associated promoter-1 (LAP1), Simian virus 40 promoter, human elongation factor 1 alpha promoter and the human muscle cell specific desmin promoter.

15. The method of claim 1, wherein the polyadenylation signal is selected from the group consisting of rabbit beta-globin poly(A) signal, synthetic polyA, HSV Thymidine kinase poly A, Human alpha globin poly A, SV40 poly A, human beta globin poly A, polyomavirus poly A, and Bovine growth hormone poly A.

16. The method of claim 1 , wherein the polypeptide composition comprises one or more HIV polypeptides selected from the group consisting of Env, Gag, Tat, Pol, Rev, Vpr and Vpu.

17. The method of claim 1, wherein the polypeptide composition comprises one or more HIV polypeptide derived epitopes comprising an Env TH epitope, an Env CTL epitope, a Gag TH epitope, a Gag CTL epitope and a Nef CTL epitope.

18. The method of claim 15, wherein the HIV polypeptide derived epitopes comprise one or more amino acid sequences of SEQ ID NO: 1 through SEQ ID NO: 8.

19. The method of claim 1, wherein component (i) further comprises a nucleotide sequence encoding an IL-12 polypeptide, wherein the IL-12 polypeptide is comprised of a p40 subunit and a p35 subunit.

20. The method of claim 19, wherein the nucleotide sequence encoding the IL-12 polypeptide is a first nucleotide sequence encoding the IL-12 p40 subunit and a second nucleotide sequence encoding the IL-12 p35 subunit, wherein the first and second nucleotide sequences are under the control of separate promoters.

21. The method of claim 19, wherein the nucleotide sequence encoding the IL-12 polypeptide is comprised in a separate DNA plasmid than the DNA plasmid encoding the HIV polypeptide.

22. The method of claim 19, wherein the nucleotide sequence encoding the IL-12 polypeptide is comprised in the same plasmid as the DNA plasmid encoding an HIV polypeptide.

23. The method of claim 1 , wherein component (i) is formulated in a citrate buffer comprising bupivicaine, sodium chloride and a chelating agent, wherein the pH of the buffer is between 6.5 and 7.2.

24. The method of claim 1 , wherein component (ii) is formulated with an adjuvant.

25. The method of claim 24, wherein the adjuvant is RC529-SE, GMCSF, or a combination thereof.

26. The method of claim 1 , wherein components (i) and (ii) are administered parenterally or mucosally.

27. The method of claim 26, wherein parenteral administration is by intramuscular injection.

28. The method of claim 27, wherein components (i) and (ii) are injected at the same intramuscular injection site.

26. The method of claim 1, wherein components (i) and (ii) are co-formulated as a single injection dosage comprising both components (i) and (ii).

27. The method of claim 1 , wherein component (i) further comprises a nucleotide sequence encoding an IL-15 polypeptide.