USE OF NON-SUBTYPE B GAG PROTEINS FOR LENTIVIRAL PACKAGING

Abstract

The invention encompasses a lentiviral packaging vector comprising a non-subtype B gag-pol sequence, particularly a subtype D gag-pol sequence. The invention further encompasses methods for making and using these vectors. The invention further encompasses lentiviral vector particles comprising HIV-1 non-subtype B Gag and/or Pol proteins.

Description

BACKGROUND OF THE INVENTION

[0001] Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection, in order to develop a specific immune response in their host.

[0002] Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufacture of inactivated vaccines.

[0003] Gene delivery using modified retroviruses (retroviral vectors) was introduced in the early 1980s by Mann et al. (Cell, 33(1):153-9, 1983). The most commonly used oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(11):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences generally required in cis are the long terminal repeats (LTRs) and its vicinity: the inverted repeats (IR or att sites) required for integration, the packaging sequence .PSI., the transport RNA-binding site (primer binding site, PBS), and some additional sequences involved in reverse transcription (the repeat R within the LTRs, and the polypurine tracts, PPT, necessary for plus strand initiation). To generate replication-defective retroviral vectors, the gag, pol, and env genes are generally entirely deleted and replaced with an expression cassette.

[0004] Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through the endogenous pathway.

[0005] Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeflciency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defenses.

[0006] The design of recombinant lentiviral vectors is based on the separation of the cis- and trans-acting sequences of the lentivirus. Efficient integration and replication in non-dividing cells requires the presence of two cis-acting sequences in the lentiviral genome, the central polypurine tract (cPPT) and the central terminal sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA "flap", which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2) 173-85; Arhel et al., 2007, EMBO J, 26(12):3025-37).

[0007] HIV-1 lentiviral vectors have been generated based on providing the subtype B Gag, Pol, Tat and Rev proteins for packaging vectors in trans from a packaging construct (Naldini et al, PNAS 15: 11382-8 (1996); Zufferey et al, Nature Biotechnology 15:871-875, 1997); Dull et al, Journal of Virology (1997)). These studies were performed with subtype B Gag and Pol proteins. The effect of non-subtype B gag and poi sequences in a HIV-1 packaging construct was not assessed.

[0008] There are many different subtypes of HIV-1 other than subtype B. Some subtypes of HIV-1, such as C, E, and A, appear to be transmitted more efficiently than HIV-1 subtype B, which is the major subtype in the United States and Europe. Essex et al., Adv Virus Res. 1999; 53:71-88. The predominant subtype of HIV-1 that is found in the developed Western World, clade B, differs considerably from those subtypes and recombinants that exist in Africa and Asia, where the vast majority of HIV-infected persons reside. Spira et al., J. Antimicrobial Chemotherapy (2003) 51, 229-240. Thus, serious discrepancies may exist between the subtype B retrovirus encountered in North America and Europe and those viral subtypes that plague humanity on a global scale. Id. Subtype diversity may impact on modes of HIV transmission. Homosexual and intravenous drug abuse are the primary modes of transmission observed for clade B strains in Europe and the Americas. Id. In contrast, clades A, C, D and E predominate in Africa and Asia where heterosexual transmission predominates. Id. In addition, some studies suggest that AIDS progression differs as a function of infecting subtype. Id. Thus, it appears that HIV-1 subtype B is quite different than the other HIV-1 subtypes.

[0009] HIV phylogenic classifications are normally based either on nucleotide sequences derived from multiple sub genomic regions (gag, pol and env) of the same isolates, or on full-length genome sequence analysis. A phylogenic analysis of HIV-1 near-full length sequences revealed that HIV-1 subtype B was most closely related genetically to HIV subtype D (FIG. 1). Phylogenic analyses of HIV-1 Gag and Pol protein sequences also showed that HIV-1 subtype B was most closely related genetically to HIV subtype D (FIG. 2).

[0010] Nevertheless, HIV-1-NDK, a subtype D virus, is significantly more cytopathic for CD4+ lymphocytes than the HIV-1-BRU prototype, a subtype B virus. This may be due to enhanced fusogenicity and infectivity of subtype D viruses. De Mareuil et al., J. Virol. 66: 6797 (1992). Phenotypic analysis of recombinant viruses indicated that 75 amino acids from the N-terminal part of HIV-1-NDK matrix (MA) protein, together with the HIV-1-NDK envelope glycoprotein, are responsible for enhanced fusogenicity of HIV-1-NDK in CD4+ lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4- cell lines. Id.

[0011] There is a need in the art for lentiviral packaging constructs producing higher titers of packaged lentiviral vectors, in order to reduce injection volumes, increase dosages, reduce the cost of vaccination, and increase the number of patients that could be treated with one batch. The current invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

[0012] The gag-pol gene of subtype B of HIV-1 in a lentiviral packaging plasmid (construct p8.74) was replaced by the gag-pol gene of a subtype D HIV-1 to generate construct pThV-GP-N. The constructs were used for lentiviral vector production. Approximately 2-fold higher titers were obtained using the pThV-GP-N plasmid as compared to construct p8.74. Thus, the Gag-Pol of a subtype D virus increases the titer of lentiviral vector particles relative to a Gag-Pol of a subtype B virus.

[0013] The invention encompasses a lentiviral packaging vector comprising a subtype D gag-pol sequence, particularly from HIV-1 NDK. In a preferred embodiment, the lentiviral packaging vector comprises the nucleotide sequence of SEQ ID NO:1. In a preferred embodiment, the lentiviral packaging vector encodes the amino acid sequence of SEQ ID NO:2.

The nucleotide sequence of SEQ ID NO 1 is:

TABLE-US-00001 (SEQ ID NO 1) atgggtgcgagagcgtcagtattaagcgggggaaaattagatacatgggaaagaattcggttacggcca ggaggaaagaaaaaatatgcactaaaacatttgatatgggcaagcagggagctagaacgatttacactt aatcctggccttttagagacatcagaaggctgtaaacaaataataggacagctacaaccatctattcaa acaggatcagaagaaattagatcattatataatacagtagcaaccctctattgtgtacatgaaaggata gaggtaaaagacaccaaagaagctgtagaaaagatggaggaagaacaaaacaaaagtaagaaaaagaca cagcaagcagcagctgatagcagccaggtcagccaaaattaccctatagtgcagaacctacaggggcaa atggtacatcaggccatatcacctagaactttgaacgcatgggtaaaagtaatagaagaaaaggccttc agcccggaagtaatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacaccatg ctaaacacagtggggggacatcaagcagctatgcaaatgctaaaagagaccatcaatgacgaagctgca gaatgggacagattacatccagtgcatgcagggcctgttgcaccaggccaaatgagagaaccaagggga agtgatatagcaggaactactagtacccttcaggaacaaatagcatggatgacaagcaacccacctatc ccagtaggagaaatctataaaagatggataatcctgggattaaataaaatagtaagaatgtatagccct gtcagcattttggacataagacagggaccaaaggaaccttttagagactatgtagaccggttctataaa actctaagagccgagcaagcttcacaggatgtaaaaaactggatgacagaaaccttgttggtccaaaat gcaaacccagattgtaaaactatcttaaaagcattgggaccacaggctacactagaagaaatgatgaca gcatgccagggagtgggggggcccggccataaagcaagagttttggctgaggcaatgagccaagtaaca ggttcagctactgcagtaatgatgcagagaggcaattttaagggcccaagaaaaagtattaagtgtttc aactgtggcaaggaagggcacacagcaaaaaattgcagggcccctagaaaaaagggctgttggaaatgc ggaagggaaggacaccaaatgaaagattgcactgaaagacaggctaattttttagggaagatttggcct tcccacaagggaaggccggggaattttcttcagagcagaccagagccaacagccccaccagcagagagc ttcgggtttggggaggagataaccccctctcagaaacaggagcagaaagacaaggaactgtatccttta gcttccctcaaatcactctttggcaacgacccctcgtcacaataaagatagggggacagctaaaggaag ctctattagatacaggagcagatgatacagtattagaagaaataaatttgccaggaaaatggaagccaa aaatgatagggggaattggaggttttatcaaagtaagacagtatgatcaaatactcatagaaatctgtg gatataaagctatgggtacagtattagtaggacctacacctgtcaacataattggaagaaatttgttga cccagattggctgcactttaaattttccaattagtcctattgaaactgtaccagtaaaattaaagccag gaatggatggcccaaaagttaaacaatggccattgacgaagaaaaaataaaagcattaacagaaatttg tacagaaatggaaaaggaaggaaaaatttcaagaattgggcctgaaaatccatataatactccaatatt tgccataaagaaaaaagacagtaccaagtggagaaaattagtagatttcagagaacttaataagagaac tcaagatttctgggaggttcaattaggaataccgcatcctgcagggctgaaaaagaaaaaatcagtaac agtactggatgtgggtgatgcatatttctcagttcccttagatgaagattttaggaaatataccgcatt taccatacctagtataaacaatgagacaccagggattagatatcagtacaatgtgctcccacagggatg gaaaggatcaccggcaatattccaaagtagcatgacaaaaatcttagagccctttagaaaacaaaatcc agaaatagttatctatcaatacatggatgatttgtatgtaggatctgacttagaaatagggcagcatag aacaaaaatagaggaattaagagaacatctattgaggtggggatttaccacaccagataaaaaacatca gaaagaacctccatttctttggatgggttatgaactccatcctgataaatggacagtacagcctataaa cctgccagaaaaagaaagctggactgtcaatgatatacagaagttagtggggaaattaaactgggcaag ccagatttatgcaggaattaaagtaaagcaattatgtaaactccttaggggaaccaaagcactaacaga agtagtaccactaacagaagaagcagaattagaactggcagaaaacagggaaattctaaaagaaccagt acatggagtgtattatgacccatcaaaagacttaatagcagaactacagaaacaaggggacggccaatg gacataccaaatttatcaagaaccatttaaaaatctaaaaacaggaaagtatgcaagaacgaggggtgc ccacactaatgatgtaaaacaattaacagaggcagtgcaaaaaatagccacagaaagcatagtgatatg gggaaagactcctaaatttaaactacccatacaaaaggaaacatgggaaacatggtggatagagtattg gcaagccacctggattcctgagtgggaatttgtcaatacccctcctttagtaaaattatggtaccagtt agagaaggaacccataataggagcagaaactttctatgtagatggggcagctaatagagagactaaatt aggaaaagcaggatatgttactgacagaggaagacagaaagttgtccctttcactgacacgacaaatca gaagactgagttacaagcaattaatctagctttacaggattcgggattagaagtaaacatagtaacaga ttcacaatatgcactaggaatcattcaagcacaaccagataagagtgaatcagagttagtcagtcaaat aatagagcagctaataaaaaaggaaaaggtttacctggcatgggtaccagcacacaaaggaattggagg aaatgaacaagtagataaattagtcagtcagggaatcaggaaagtactatttttggatggaatagataa ggctcaggaagaacatgagaaatatcacaacaattggagagcaatggctagtgattttaacctaccacc tgtggtagcgaaagaaatagtagctagctgtgataaatgtcagctaaaaggagaagccatgcatggaca agtagactgtagtccaggaatatggcaattagattgtacacatctggaaggaaaagttatcctggtagc agttcatgtagccagtggctatatagaagcagaagttattccagcagaaacggggcaagaaacagcata ctttctcttaaaattagcaggaagatggccagtaaaagtagtacatacagataatggcagcaatttcac cagtgctacagttaaggccgcctgttggtgggcagggatcaaacaggaatttggaattccctacaatcc ccaaagtcaaggagtagtagaatctatgaataaagaattaaagaaaattataggacaggtaagagatca agctgaacatcttaagacagcagtacaaatggcagtatttatccacaattttaaaagaaaaggggggat tgggggatacagtgcaggggaaagaataatagacataatagcaacagacatacaaactagagaattaca aaaacaaatcataaaaattcaaaattttcgggtttattacagggacagcagagatccaatttggaaagg accagcaaagcttctctggaaaggtgaaggggcagtagtaatacaagacaatagtgacataaaggtagt accaagaagaaaagtaaagatcattagggattatggaaaacagatggcaggtgatgattgtgtggcaag tagacaggatgaggattaac.

[0014] The amino acid sequence of SEQ ID NO 2 is:

TABLE-US-00002 (SEQ ID NO: 2) MGARASVLSGGKLDAWERIRLRPGGKKKYALKHLIWASRELERIALN PGLLETSEGCKQIIGQLQPSIQTGSEELRSLYNTIATLYCVHERIEV KDTKEAVEKMEEEQNKSKKKTQQAAADSSQVSQNYPIVQNLQGQMVH QAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTV GGHQAAMQMLKETINDEAAEWDRLHPVHAGPVAPGQMREPRGSDIAG TTSTLQEQIAWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDI RQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKT ILKALGPQATLEEMMTACQGVGGPGHKARVLAEAMSQVTGSVTAVMM QRGNFKGPRKSIKCFNCGKEGHTAKNCRAPRKKGCWKCGREGHQMKD CSERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFGFGEEITPS QKQEQKDKELYPLASLKSLFGNDPSSQFFREDLAFPQGKAGEFSSEQ TRANSPTSRELRVWGGDNPLSETGAEGQGTVSFSFPQITLWQRPLVT IKIGGQLKEALLDTGADDTVLEEMNLPGKWKPKMIGGIGGFIKVRQY DQILIEICGYKAMGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIET VPVKLKPGMDGPKVKQWPLTEEKIKALTEICTEMEKEGKISRIGPEN PYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLK KKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNV LPQGWKGSPAIFQSSMTKILEPFRKQNPEIVIYQYMDDLYVGSDLEI GQHRTKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQ PIKLPEKESWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKAL TEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAELQKQGDGQ WTYQIYQEPFKNLKTGKYARTRGAHTNDVKQLTEAVQKIATESIVIW GKTPKFKLPIQKETWETWWIEYWQATWIPEWEFVNTPPLVKLWYQLE KEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPFTDTTNQKT ELQAINLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQ LIKKEKVYLAWVPAHKGIGGNEQVDKLVSQGIRKVLFLDGIDKAQEE HEKYHNNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHGQVDCSP GIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLA GRWPVKVVHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVE SMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGER IIDIIATDIQTRELQKQIIKIQNFRVYYRDSRDPIWKGPAKLIWKGE GAVVIQDNSDIKVVPRRKVKIIRDYGKQMAGDDCVASRQDED.

[0015] The invention encompasses a lentiviral packaging vector comprising a subtype D MA sequence, particularly from HIV-1 NDK. In a preferred embodiment, the lentiviral packaging vector encodes an MA protein comprising the amino acid sequence of SEQ ID NO:3.

[0016] The amino acid sequence of SEQ ID NO 3 is:

TABLE-US-00003 (SEQ ID NO: 3) MGARASVLSGGKLDTWERIRLRPGGKKKYALKHLIWASRELERFTLNPG LLETSEGCKQIIGQLQPSIQTGSEEIRSLYNTVATLYCVHERIEVKDTK EAVEKMEEEQNKSKKKTQQAAADSSQVSQNY.

[0017] Preferably, the lentiviral packaging vector encoding the HIV Gag MA protein generates at least a 1.5 fold increase, or at least a 2-fold increase, in the titer of a packaged lentiviral vector as compared to the lentiviral packaging vector encoding an HIV Gag MA protein of HIV-1 BRU, e.g., p8.74. Preferably, the lentiviral packaging vector is replication-defective and lacks a .PSI. site.

[0018] In a preferred embodiment, the lentiviral packaging vector encodes an HIV Gag MA protein having an amino acid at position 12 that is not a glutamic acid and an amino acid at position 15 that is not an arginine. Preferably, the lentiviral packaging vector does not have both a valine at position 46 and a leucine at position 61.

[0019] In a preferred embodiment, the amino acid at position 12 of the MA protein is a lysine. In a preferred embodiment, the amino acid at position 15 is a threonine. In a preferred embodiment, the amino acid at position 15 is an alanine. In a preferred embodiment, the amino acid at position 46 is a leucine. In a preferred embodiment, the amino acid at position 61 is an isoleucine. In a preferred embodiment, the amino acid at position 61 is a methionine.

[0020] In one embodiment, the vector does not encode a functional Env protein.

[0021] The invention also encompasses methods for making the above lentiviral packaging vectors and methods for using these lentiviral packaging vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The invention is more fully understood through reference to the drawings.

[0023] FIG. 1 depicts phylogenic trees of HIV viruses.

[0024] FIGS. 2A and B depict phylogenic trees of HIV GAG (A) and POL (B) proteins.

[0025] GAG and POL protein sequences were obtained from: http://www.hiv.lanl.gov/content/sequence/NEWALGN/align.html. For each known HIV clade, one patient's (for clade B) or two patients' virus sequences were randomly chosen and GAG and POL protein sequences were compared to the reference clade B proteins (B.FR.83.HXB2_LAI_IIIB_BRU.KO3455). Alignments were performed using Vector NTI advance 11 (Invitrogen).

[0026] FIG. 3 depicts titers obtained using the p8.74 and the pThV-GP-N plasmids for vector production. Lentiviral particles were produced using the proviral plasmid (pFLAP-CMV-GFP), the pseudotyping plasmid (pTHV-VSV.G) and either the commonly used packaging plasmid (derived from the BRU strain, p8,74) or an NDK-derived packaging plasmid (pTHV-GP-N). With each packaging plasmid, 18 independent transfections were performed and the particles titers were measured by FACS analysis. Similar results were also obtained using a vector employing a proviral plasmid containing the 02 microglobulin promoter driving HIV antigen expression.

[0027] FIGS. 4A and B depicts production of wild type BRU and NDK viruses and evaluation of their respective early phase efficiency. 293T cells were transfected either with plasmid encoding for the wild type BRU (pBRU) or NDK (pNDK-N) virus. Viral supernatants were collected after 48 hours and diluted to infect P4-CCR5 cells (encompassing a stable luciferase reporting gene which expression is driven by the HIV LTR, allowing a luciferase production in presence of the TAT protein (brought by the virus). Serial dilution of either BRU or NDK viruses were used to infect P4-CCR5 cells and the luciferase expression (A) or the luciferase/P24 ratio (B) were measured.

[0028] FIG. 5 depicts vector production using different ratios of BRU (p8,74) and NDK (pThV GP-N) derived packaging plasmids. For each conditions (from 0 .mu.g NDK+10 .mu.g BRU to 10 .mu.g NDK+0 .mu.g BRU), the titer (gray bars) and the P24 level (black squares) were measured.

[0029] FIG. 6 depicts a Western blot of vector supernatants produced using either BRU (8,74) or NDK (pThV GP-N) derived packaging plasmids. The P24 protein and precursors detection was performed using the NIH anti-P24 MAB (183-H12-5C).

[0030] FIG. 7 depicts a sequence alignment of the N-terminal MA sequences of a clade B virus (BRU; SEQ ID NO:3) with a clade D virus (NDK; SEQ ID NO:4).

[0031] FIG. 8 depicts a sequence alignment of the N-terminal MA sequences of clade B viruses.

[0032] FIG. 9 depicts a sequence alignment of the N-terminal MA sequences of clade D viruses.

[0033] FIG. 10 depicts a sequence alignment of the N-terminal MA sequences of a clade B virus (BRU) with viruses of other clades.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Subtype B HIV-1 viruses differ from other HIV-1 subtypes in that subtype B viruses appear to be transmitted less efficiently and have a different mode of transmission. Nevertheless, subtype B viruses have been used extensively in the generation of HIV-1 lentiviral vectors and lentiviral packaging vectors. To determine whether the Gag and Pol proteins of non-subtype B viruses could be used for the generation of lentiviral packaging vectors, the gag-pol gene of HIV-1 subtype B in a lentiviral packaging plasmid (construct p8.74) was replaced by the gag-pol gene of a subtype D HIV-1 to generate construct pThV-GP-N. The constructs were used for lentiviral vector production. Approximately 2-fold higher titers were obtained using the pThV-GP-N plasmid as compared to construct p8.74 (FIG. 3). Thus, the subtype D HIV-1 Gag-Pol in the packaging vector increased the titer of lentiviral vector over the titer seen with a subtype B HIV-1 Gag-Pol.

[0035] Increased titer of the lentiviral vector is beneficial in allowing the reduction in contaminants in a given dose of lentiviral vector. This facilitates a reduction in injection volumes, and an increase in possible dosages. It further facilitates reducing the cost of vaccination by reducing the quantity of materials and labor necessary to achieve a particular dose, and by increasing the number of patients that could be treated with a single batch of lentival vector.

[0036] Serial dilutions of HIV-1 BRU and HIV-1 NDK viruses indicated that the HIV-1 NDK virus was more efficient in the early phases of the virus life cycle (FIG. 4). By mixing different amounts of the subtype B and subtype D packaging vectors, it was demonstrated that the subtype D packaging vector increased both the titer and the level of p24 in the lentiviral vector preparations (FIG. 5). The p24 in the lentiviral vector preparations using the subtype D packaging vector was also observed to be less completely processed, showing higher levels of p24 precursors (FIG. 6). Thus, the Gag protein in the subtype D packaging vector was exhibiting various differences from the subtype B packaging vectors.

[0037] It is known that, with HIV-1 Env, the 75 amino acids from the N-terminal part of HIV-1-NDK matrix (MA) protein is responsible for enhanced fusogenicity of HIV-1-NDK in CD4+ lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4- cell lines. De Mareuil et al., J. Virol. 66: 6797 (1992). Since the Env protein used for generation of lentiviral vectors with the subtype B and subtype D packaging vectors is the same (i.e., VSV), only the differences in HIV-1 MA are present in the subtype B and subtype D packaging vectors.

[0038] The conservation and divergence of amino acids in the N-terminal 75 amino acids of M from various clades of HIV-1 viruses was examined. HIV-1 NDK showed 10 differences in amino acids from HIV-1 BRU (FIG. 7). However, only eight of these differences were seen when HIV-1 NDK was compared to an assortment of 20 different HIV-1 subtype B viruses (FIG. 8). When other subtype D viruses were included in the comparison, only 4 of these differences remained (FIG. 9). These differences are the absence of a glutamic acid at amino acid position 12, the absence of an arginine at amino acid position 15, the absence of a valine at amino acid position 46, and the absence of a leucine at amino acid position 61 in subtype D viruses.

[0039] When other subtype viruses were included in the comparison, the other subtypes appeared to align with subtype D, preserving nearly all of these differences (FIG. 10). Nearly all of the non-subtype B viruses exhibited a lysine at position 12. Many of the non-subtype B viruses exhibited an alanine at position 15. Nearly all of the non-subtype B viruses exhibited a leucine at position 46. Nearly all of the non-subtype B viruses exhibited a methionine or isoleucine at position 61. The non-subtype B viruses exhibited the consensus of a lysine at position 12, an amino acid other than arginine at position 15, a leucine at position 46, and an isoleucine or methionine at position 61.

[0040] The invention encompasses packaging vectors encoding non-subtype B Gag and/or Pol proteins and host cells comprising these vectors. The invention further encompasses methods for making packaging vectors encoding non-subtype B Gag and/or Pol proteins. The invention also encompasses methods for using these packaging vectors to generate lentiviral vectors, and lentiviral vectors comprising non-subtype B Gag and/or Pol proteins.

[0041] Packaging Vectors

[0042] The invention encompasses packaging vectors encoding non-subtype B Gag and/or Pol proteins. A lentiviral "packaging vector" is defined herein as a nucleic acid sequence not encoding functional HIV-1 Env and lacking a .PSI. site, but capable of expressing lentiviral Gag and/or Pol proteins that can be incorporated into viral particles when cotransfected with a vector containing appropriate lentiviral cis-acting signals for packaging. The lentiviral packaging vector of the invention is unable to replicate itself by packaging and reverse transcribing its own sequence.

[0043] The packaging vector can be an RNA or DNA vector. The non-subtype B Gag and Pol proteins can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof. A preferred packaging vector comprises SEQ ID NO:1 or encodes SEQ ID NO:2.

[0044] The invention encompasses packaging vectors encoding non-subtype B Gag proteins and host cells comprising these vectors. The non-subtype B Gag proteins can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof. A preferred packaging vector encodes the Gag protein portion of SEQ ID NO:2.

[0045] The invention encompasses packaging vectors encoding non-subtype B MA proteins and host cells comprising these vectors. The non-subtype B MA proteins can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof. A preferred packaging vector encodes SEQ ID NO:3 or the MA protein portion of SEQ ID NO:2.

[0046] In various embodiments, the packaging vector comprises SEQ ID NO:1 or a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical with SEQ ID NO:1. In various embodiments, the packaging vector encodes SEQ ID NO:2, SEQ ID NO:3, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical with SEQ ID NO:2 or SEQ ID NO:3.

[0047] As used herein, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG), using the default parameters for the GAP program including: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0048] In various embodiments, the vector comprises a sequence encoding an HIV-1 MA protein having one or more of the following features:

[0049] the absence of a glutamic acid at amino acid position 12;

[0050] the absence of an arginine at amino acid position 15;

[0051] the absence of a valine at amino acid position 46; and

[0052] the absence of a leucine at amino acid position 61.

[0053] In various embodiments, the vector comprises a sequence encoding an HIV-1 MA protein having one or more of the following features:

[0054] the amino acid at position 12 is a lysine;

[0055] the amino acid at position 15 is a threonine;

[0056] the amino acid at position 15 is an alanine;

[0057] the amino acid at position 46 is a leucine;

[0058] the amino acid at position 61 is an isoleucine; and/or

[0059] the amino acid at position 61 is a methionine.

[0060] The packaging vector preferably encodes HIV-1 Gag and Pol. Most preferably, the packaging vector encodes an HIV-1 Gag MA protein.

[0061] The packaging vector can contain viral or non-viral sequences for expression of Gag and Pol. The packaging vector can contain an HIV-1 LTR or the U3 region of an HIV-1 LTR. In other embodiments, the packaging vector does not contain HIV-1 LTRs. Any promoter can be used to drive expression of Gag and Pol. Preferably, the promoter is a strong promoter in human cells. Most preferably, the packaging vector contains a Cytomegalovirus (CMV) promoter, a CMV early enhancer/chicken 1 actin (CAG) promoter, a Rous Sarcoma Virus (RSV) promoter, a human phosphoglycerate kinase (hPGK) promoter, or a U3 from an LTR (e.g., myeloproliferative sarcoma virus (MPSV) U3) promoter driving expression of the encoded genes, e.g. gag and pol.

[0062] Preferably, the packaging vector contains a polyadenylation signal. Any polyadenylation signal can be. Preferably, polyadenylation signal is a strong signal in human cells. Most preferably, the polyadenylation signal is a human .alpha.2 globin or a Bovine Growth hormone (BGH) polyadenylation signal.

[0063] Preferably, the packaging vector contains a Rev-responsive element (RRE). In a preferred embodiment, the packaging vector expresses an HIV-1 Rev protein. In a preferred embodiment, the packaging vector contains at least one splice donor site and at least one splice acceptor site. In one embodiment, the packaging vector expresses an HIV-1 Tat protein.

[0064] In preferred embodiments, the packaging vector lacks sequences encoding HIV-1 Vif, Vpr, Vpu, and/or Nef. The packaging vector can comprise a sequence encoding an HIV-1 MA protein having one or more of the features discussed herein.

[0065] In one embodiment, the packaging vector encodes only 1 HIV-1 protein, Gag. In one embodiment, the packaging vector encodes only 2 HIV-1 proteins, Gag and Pol. In one embodiment, the packaging vector encodes only 3 HIV-1 proteins, selected from Gag, Pol, Rev, and Tat. In one embodiment, the packaging vector encodes only 4 HIV-1 proteins, Gag, Pol, Rev, and Tat.

[0066] In one embodiment, the vector comprises (from 5' to 3') a CMV promoter, a nucleic acid sequence encoding HIV-1 Gag-Pol, an exon encoding part of Tat and Rev, a splice donor site, an intron containing an RRE, a splice acceptor site, an exon encoding part of Tat and Rev, and a polyadenylation signal. Preferably, the HIV-1 Gag-Pol is a subtype D HIV-1 Gag-Pol.

[0067] The packaging vector may further contain an origin for replication in bacteria or eukaryotic cells. The packaging vector may contain a selectable marker gene for selection in bacteria or eukaryotic cells.

[0068] The invention encompasses host cells comprising the packaging vectors of the invention. The host cells can be transiently transfected with the packaging vectors of the invention. The host cells can be cell lines with the packaging vectors of the invention integrated into the genome of the host cell. Many different cells are suitable host cells Preferably, the cells are human cells, most preferably an immortalized human cell line. In one embodiment, the cells are HEK 293T cells. In one embodiment, the cells are HeLA, HT1080 or PER C6 cells (Delenda et al, Cells for Gene Therapy and vector Production, from Methods in Biotechnology, Vol 24: Animal Cell Biotechnology: Methods and Protocols, 2.sup.nd Ed. Edited by R. Portner, Humana Press Inc., Totowa, N.J.).

[0069] Packaging Systems

[0070] The invention encompasses lentiviral packaging systems comprising cells expressing non-subtype B Gag and/or Pol proteins. A lentiviral "packaging system" is defined herein as a cell-based system comprising cells expressing at least lentiviral Gag and Pol proteins in the absence of a .PSI. site, and capable of packaging and reverse transcribing an exogenous nucleic acid containing a .PSI. site. The cells of the lentiviral packaging system can also express other viral proteins. Preferably, the lentiviral packaging system expresses an envelope protein. The envelope protein can be a lentiviral (e.g., HIV-1 Env) or non-lentiviral (e.g., VSV, Sindbis virus, Rabies virus) envelope protein. In various embodiments, the lentiviral packaging system expresses an HIV-1 Tat and/or Rev protein.

[0071] In various embodiments, the cells of the lentiviral packaging system contain sequences encoding HIV-1 Gag and/or Pol stably integrated into their genome. In various embodiments, the cells of the lentiviral packaging system contain sequences encoding an envelope protein stably integrated into their genome. In various embodiments, the cells of the lentiviral packaging system contain sequences encoding HIV-1 Tat and/or Rev stably integrated into their genome.

[0072] In various embodiments, the cells of the lentiviral packaging system transiently express HIV-1 Gag and/or Pol proteins. In various embodiments, the cells of the lentiviral packaging system transiently express an envelope protein. In various embodiments, the cells of the lentiviral packaging system transiently express HIV-1 Tat and/or Rev proteins.

[0073] The cells of the lentiviral packaging system can express non-subtype B Gag and Pol proteins selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof. In one embodiment, the cells of the lentiviral packaging system comprise SEQ ID NO:1 or express SEQ ID NO:2.

[0074] In various embodiments, the cells of the lentiviral packaging system express non-subtype B Gag proteins. The non-subtype B Gag proteins can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof.

[0075] In various embodiments, the cells of the lentiviral packaging system express non-subtype B MA proteins. The non-subtype B MA proteins can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J proteins, and recombinants thereof. A preferred packaging vector encodes SEQ ID NO:3 or the MA protein portion of SEQ ID NO:2.

[0076] In various embodiments, the cells of the lentiviral packaging system can contain any of the lentiviral vectors of the invention.

[0077] In various embodiments, the cells of the lentiviral packaging system contain SEQ ID NO:1 or a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical with SEQ ID NO:1. In various embodiments, the cells of the lentiviral packaging system express a protein with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical with SEQ ID NO:2 or SEQ ID NO:3.

[0078] Methods of Producing Packaging Vectors

[0079] The invention encompasses methods for making packaging vectors encoding non-subtype B Gag and/or Pol proteins. The packaging vector can comprise any of the features discussed herein.

[0080] In one embodiment, the method comprises inserting a Gag and/or Pol sequence from a non-subtype B HIV-1 virus into a plasmid under the control of a non-HIV promoter (e.g., CMV promoter) to generate a packaging vector. In one embodiment, the packaging vector encodes only 1 HIV-1 protein. In one embodiment, the packaging vector encodes only 2 HIV-1 proteins. In one embodiment, the packaging vector encodes only 3 HIV-1 proteins, selected from Gag, Pol, Rev, and Tat. In one embodiment, the packaging vector encodes only 4 HIV-1 proteins, Gag, Pol, Rev, and Tat.

[0081] In various embodiments, the plasmid comprises one or more of a CMV promoter, an exon encoding part of Tat and Rev, a splice donor site, an intron containing an RRE, a splice acceptor site, an exon encoding part of Tat and Rev, and a polyadenylation signal.

[0082] In various embodiments, the packaging vector comprises a CMV promoter, an exon encoding part of Tat and Rev, a splice donor site, an intron containing an RRE, a splice acceptor site, an exon encoding part of Tat and Rev, and a polyadenylation signal.

[0083] The non-subtype B HIV-1 virus can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J viruses, and recombinants thereof.

[0084] In one embodiment, the method comprises providing a packaging vector comprising a Gag sequence of an HIV-1 subtype B virus and replacing Gag sequences in the vector with sequences from an HIV-1 non-subtype B virus.

[0085] The non-subtype B HIV-1 virus can be selected from subtype A, subtype C, subtype D, subtype E, subtype F1, subtype F2, subtype G, subtype H, and subtype J viruses, and recombinants thereof. In a preferred embodiment, non-subtype B HIV-1 virus is HIV-1 NDK.

[0086] Methods of Producing Lentiviral Vectors

[0087] The invention also encompasses methods for using packaging vectors encoding HIV-1 non-subtype B Gag and/or Pol proteins to generate lentiviral vectors. In one embodiment, the invention encompasses administering a packaging vector encoding an HIV-1 non-subtype B Gag or Pol protein to a cell with a lentiviral vector. The packaging vector can comprise any of the features discussed herein.

[0088] The lentiviral vector comprises cis-acting sequences for packaging and reverse transcription, including a .PSI. site and primer binding site. Preferably, the lentiviral vector comprises two HIV-1 LTR sequences. In one embodiment, one of the LTRs is deleted for U3 and R sequences. Preferably, the lentiviral vector comprises a central polypurine tract (cPPT) and a central terminal sequence (CTS). The lentiviral vector preferably encodes a lentiviral or non-lentiviral protein, such as a selectable marker or tumor antigen.

[0089] In one embodiment, the lentiviral vector comprises one or more HIV antigen, preferably an HIV-1 antigen. Most preferably, the antigen is a Gag, Pol, Env, Vif, Vpr, Vpu, Nef, Tat, or Rev antigen. The antigen can be a single antigen, a mix of antigens, an antigenic polypeptide, or a mix of antigenic polypeptides from these proteins. In a preferred embodiment, the lentiviral vector comprises an HIV-1 p24 Gag antigen.

[0090] In one embodiment, the invention encompasses a lentiviral vector comprising an NIS-containing promoter. An "NIS-containing promoter" comprises an NF-Kb binding site, an interferon stimulated response element (ISRE), and an SXY module (SXY). Examples of naturally occurring NIS-containing promoters are the .beta.2m promoter and the MHC class I gene promoters. These naturally occurring NIS-containing promoters are generally cloned or reproduced from the promoter region of a gene encoding a protein .beta.2m or a MHC class I protein, or referred to as putatively encoding such proteins in genome databases (ex: NCBI polynucleotide database http://www.ncbi.nlm.nih.gov/guide/dna-ma). Both .beta.2m and class I MHC proteins enter the Major Histocompatibility Complex (MHC). .beta.2m and class I MHC promoter sequences are also usually referred to as such in genome databases--i.e. annotated as being .beta.2m and class I MHC promoter sequences.

[0091] MHC class I and .beta.2-microglobulin promoters contain the shared structural homologies of NIS-containing promoters. These promoters also share the ability to be strongly activated in dendritic cells, as well as, to lower intensity, in the majority of the other human body tissues.

[0092] In one embodiment, the packaging vector and the lentiviral vector are introduced together into a cell to allow the formation of lentiviral vector particles containing the Gag protein produced by the packaging vector and the nucleic acid produced by the lentiviral vector. Preferably, this is achieved by cotransfection of the cells with the packaging vector and the lentiviral vector. The cells can also be transfected with a nucleic acid encoding an Env protein, preferably a VSV Glycoprotein G. Preferably, the lentiviral vector particles are capable of entry, reverse transcription, and expression in an appropriate host cell.

[0093] In one embodiment, the packaging vector or the lentiviral vector is stably integrated into cells, and the non-integrated vector is transfected into the cells to allow the formation of lentiviral vector particles.

[0094] In one embodiment, the method further comprises collecting the lentiviral vector produced by the cells.

[0095] In one embodiment, the method further comprises selecting for a packaging vector that packages a higher titer of the lentiviral vector than a same packaging vector encoding a subtype B Gag or Pol protein. Preferably, the titer is increased at least 1.5 or 2-fold relative to the packaging vector encoding a subtype B Gag or Pol protein.

[0096] Lentiviral Vector Particles

[0097] The invention also encompasses lentiviral vector particles comprising HIV-1 non-subtype B Gag and/or Pol proteins. The non-subtype B Gag and Pol proteins can comprise any of the features discussed herein.

[0098] The lentiviral vector particle comprises a nucleic acid comprising cis-acting sequences for packaging and reverse transcription, including a .PSI. site and primer binding site in association with Gag, Pol and Env proteins. Preferably, the nucleic acid comprises two HIV-1 LTR sequences. In one embodiment, one of the LTRs is deleted for U3 and R sequences. Preferably, the nucleic acid of the lentiviral vector particle comprises a central polypurine tract (cPPT) and a central terminal sequence (CTS). The nucleic acid preferably encodes a lentiviral or non-lentiviral protein, such as a selectable marker or tumor antigen. Preferably, the lentiviral vector particle comprises a VSV Glycoprotein.

[0099] Preferably, the lentiviral vector comprises an NIS-containing promoter. In one embodiment, the promoter is a .beta.2m promoter.

[0100] In one embodiment, the lentiviral vector comprises one or more HIV antigen, preferably an HIV-1 antigen. Most preferably, the antigen is a Gag, Pol, Env, Vif, Vpr, Vpu, Nef, Tat, or Rev antigen.

[0101] The lentiviral vectors of the invention can be administered to a host cell, including a human host.

[0102] The lentiviral vector particle can contain a targeting mechanism for specific cell types. See, e.g., Yang et al., Targeting lentiviral vectors to specific cell types in vivo, PNAS 113(31):11479-11484 (2006), which is hereby incorporated by reference. Targeting can be achieved through an antibody that binds to a cell surface protein on a cell. The targeted cell type is preferably a dendritic cell, a T cell, a B cell. Targeting to dendritic cell type is preferred and can be accomplished through envelope proteins that specifically bind to a DC surface protein. See, e.g., Yang at al., Engineered Lentivector Targeting of Dendritic Cells for In Vivo, Nat Biotechnol. 2008 March; 26(3): 326-334, which is hereby incorporated by reference.

[0103] The present invention further relates to the use of the lentiviral vectors according to the invention, especially in the form of lentiviral vector particles, for the preparation of therapeutic compositions or vaccines which are capable of inducing or contributing to the occurrence or improvement of an immunogical reaction against epitopes, more particularly those encoded by the transgene present in the vectors.

EXAMPLES

Example 1. Plasmid Construction

[0104] The gag-pol gene was amplified by PCR, using two primers and pNDK-N, a clone of HIV-1 NDK, as template. In order to obtain pThV-GP-N plasmid, the PCR product was digested with EagI/SalI and inserted in packaging construct p8.74, also digested by EagI/SalI.

Example 2. Production of Lentiviral Vector by Transfection

[0105] Lentiviral vector stock was produced using pFLAP CMV GFP bis and pTHV-VSV.G (INDI-CO)bis in combination with p8.74 or pThV-GP-N. 36 transfections were done, 18 with p8.74 and 18 with pThV-GP-N. All the supernatant were stored at -80.degree. C.

[0106] The plasmid pFLAP-CMV GFP bis encoded for the Green Fluorescence protein (GFP), which expression can be detected by flow cytometry.

Example 3. Titration of Lentiviral Vector Production

[0107] Vector titer was determined by the frequency of GFP expression in 293T cells. Cells were cultivated in 24-well plates, in DMEM containing 10% FBS, until they reached a density of 1.times.10.sup.5 cells per well. The cells were then transduced with different volume of vector supernatant in a final volume of 300 .mu.L. After 2 hours, 700 .mu.l of fresh medium containing 10% FBS was added in each well. 72 hours after transduction, the medium was then removed and the cells washed in Dulbecco's phosphate-buffered saline (DPBS; Gibco). Cells were removed with 0.05% Trypsin-EDTA (Gibco). Trypsinization was stopped by the addition of 300 .mu.l complete DMEM, and the cells were transferred to a tube for FACS, after which the number of GFP-expressing cells was counted with a FACSCalibur (BD Biosciences) using an excitation wavelength of 509 nm. Only the percent of GFP positive cells under 30% was considered.

[0108] The results are shown in FIG. 3. A significant difference between the pThV-GP-N and the p8.74 vectors productions was seen, with higher titers obtained using the pThV-GP-N plasmid. Indeed, the vector titer obtained with the packaging plasmid pThV-GP-N was 2 fold higher than the vector titer obtained with the classically used plasmid p8.74 (p<0.001 according to Student test).

Example 4. Increased Titer of Lentiviral Vector with pThV-GP-N

[0109] HIV-1 BRU and HIV-1 NDK viruses were made on 293T cells and used to transduced P4 CCR5 cells. These cells encompass a stable luciferase gene under the control of the HIV LTR: If they are transduced with TAT protein (which is the case when they are infected with a WT HIV), the LacZ gene is expressed and a luciferase expression can be measured. HIV-1 Gag p24 and luciferase expression were measured. The results are shown in FIG. 4, and confirm that wild type NDK virus has a higher transduction rate than the wild type BRU one.

Example 5. Increased p24 and Titer with pThV-GP-N

[0110] Different ratios of p8,74 and pSD GP NDK packaging vectors were used to produce lentiviral vector particles. For each ratio, the titer and the P24 level were measured. The results are shown in FIG. 5 and demonstrate that it is the presence of the NDK packaging plasmid that is responsible for the enhancement of the production titers and of the P24 level.

Example 6. Decreased p24 Processing with pThV-GP-N

[0111] Packaging vectors p8.74 and pTHV-GP-N were used to produce supernatants containing lentiviral vector particles. Western blots were performed on the supernatants using the NIH anti-P24 MAB (183-H12-5C). The results are shown in FIG. 6, and confirm that the difference between the NDK and BRU packaging plasmids relies on the production of P24 protein and precursor, as the NDK seems to generate a higher P24 synthesis (presence of P24 precursor in the viral supernatant) when the BRU shows only P24 in viral supernatant.

Sequence CWU 1

1

6714298DNAHuman immunodeficiency virus type 1 1atgggtgcga gagcgtcagt attaagcggg ggaaaattag atacatggga aagaattcgg 60ttacggccag gaggaaagaa aaaatatgca ctaaaacatt tgatatgggc aagcagggag 120ctagaacgat ttacacttaa tcctggcctt ttagagacat cagaaggctg taaacaaata 180ataggacagc tacaaccatc tattcaaaca ggatcagaag aaattagatc attatataat 240acagtagcaa ccctctattg tgtacatgaa aggatagagg taaaagacac caaagaagct 300gtagaaaaga tggaggaaga acaaaacaaa agtaagaaaa agacacagca agcagcagct 360gatagcagcc aggtcagcca aaattaccct atagtgcaga acctacaggg gcaaatggta 420catcaggcca tatcacctag aactttgaac gcatgggtaa aagtaataga agaaaaggcc 480ttcagcccgg aagtaatacc catgttttca gcattatcag aaggagccac cccacaagat 540ttaaacacca tgctaaacac agtgggggga catcaagcag ctatgcaaat gctaaaagag 600accatcaatg acgaagctgc agaatgggac agattacatc cagtgcatgc agggcctgtt 660gcaccaggcc aaatgagaga accaagggga agtgatatag caggaactac tagtaccctt 720caggaacaaa tagcatggat gacaagcaac ccacctatcc cagtaggaga aatctataaa 780agatggataa tcctgggatt aaataaaata gtaagaatgt atagccctgt cagcattttg 840gacataagac agggaccaaa ggaacctttt agagactatg tagaccggtt ctataaaact 900ctaagagccg agcaagcttc acaggatgta aaaaactgga tgacagaaac cttgttggtc 960caaaatgcaa acccagattg taaaactatc ttaaaagcat tgggaccaca ggctacacta 1020gaagaaatga tgacagcatg ccagggagtg ggggggcccg gccataaagc aagagttttg 1080gctgaggcaa tgagccaagt aacaggttca gctactgcag taatgatgca gagaggcaat 1140tttaagggcc caagaaaaag tattaagtgt ttcaactgtg gcaaggaagg gcacacagca 1200aaaaattgca gggcccctag aaaaaagggc tgttggaaat gcggaaggga aggacaccaa 1260atgaaagatt gcactgaaag acaggctaat tttttaggga agatttggcc ttcccacaag 1320ggaaggccgg ggaattttct tcagagcaga ccagagccaa cagccccacc agcagagagc 1380ttcgggtttg gggaggagat aaccccctct cagaaacagg agcagaaaga caaggaactg 1440tatcctttag cttccctcaa atcactcttt ggcaacgacc cctcgtcaca ataaagatag 1500ggggacagct aaaggaagct ctattagata caggagcaga tgatacagta ttagaagaaa 1560taaatttgcc aggaaaatgg aagccaaaaa tgataggggg aattggaggt tttatcaaag 1620taagacagta tgatcaaata ctcatagaaa tctgtggata taaagctatg ggtacagtat 1680tagtaggacc tacacctgtc aacataattg gaagaaattt gttgacccag attggctgca 1740ctttaaattt tccaattagt cctattgaaa ctgtaccagt aaaattaaag ccaggaatgg 1800atggcccaaa agttaaacaa tggccattga cgaagaaaaa ataaaagcat taacagaaat 1860ttgtacagaa atggaaaagg aaggaaaaat ttcaagaatt gggcctgaaa atccatataa 1920tactccaata tttgccataa agaaaaaaga cagtaccaag tggagaaaat tagtagattt 1980cagagaactt aataagagaa ctcaagattt ctgggaggtt caattaggaa taccgcatcc 2040tgcagggctg aaaaagaaaa aatcagtaac agtactggat gtgggtgatg catatttctc 2100agttccctta gatgaagatt ttaggaaata taccgcattt accataccta gtataaacaa 2160tgagacacca gggattagat atcagtacaa tgtgctccca cagggatgga aaggatcacc 2220ggcaatattc caaagtagca tgacaaaaat cttagagccc tttagaaaac aaaatccaga 2280aatagttatc tatcaataca tggatgattt gtatgtagga tctgacttag aaatagggca 2340gcatagaaca aaaatagagg aattaagaga acatctattg aggtggggat ttaccacacc 2400agataaaaaa catcagaaag aacctccatt tctttggatg ggttatgaac tccatcctga 2460taaatggaca gtacagccta taaacctgcc agaaaaagaa agctggactg tcaatgatat 2520acagaagtta gtggggaaat taaactgggc aagccagatt tatgcaggaa ttaaagtaaa 2580gcaattatgt aaactcctta ggggaaccaa agcactaaca gaagtagtac cactaacaga 2640agaagcagaa ttagaactgg cagaaaacag ggaaattcta aaagaaccag tacatggagt 2700gtattatgac ccatcaaaag acttaatagc agaactacag aaacaagggg acggccaatg 2760gacataccaa atttatcaag aaccatttaa aaatctaaaa acaggaaagt atgcaagaac 2820gaggggtgcc cacactaatg atgtaaaaca attaacagag gcagtgcaaa aaatagccac 2880agaaagcata gtgatatggg gaaagactcc taaatttaaa ctacccatac aaaaggaaac 2940atgggaaaca tggtggatag agtattggca agccacctgg attcctgagt gggaatttgt 3000caatacccct cctttagtaa aattatggta ccagttagag aaggaaccca taataggagc 3060agaaactttc tatgtagatg gggcagctaa tagagagact aaattaggaa aagcaggata 3120tgttactgac agaggaagac agaaagttgt ccctttcact gacacgacaa atcagaagac 3180tgagttacaa gcaattaatc tagctttaca ggattcggga ttagaagtaa acatagtaac 3240agattcacaa tatgcactag gaatcattca agcacaacca gataagagtg aatcagagtt 3300agtcagtcaa ataatagagc agctaataaa aaaggaaaag gtttacctgg catgggtacc 3360agcacacaaa ggaattggag gaaatgaaca agtagataaa ttagtcagtc agggaatcag 3420gaaagtacta tttttggatg gaatagataa ggctcaggaa gaacatgaga aatatcacaa 3480caattggaga gcaatggcta gtgattttaa cctaccacct gtggtagcga aagaaatagt 3540agctagctgt gataaatgtc agctaaaagg agaagccatg catggacaag tagactgtag 3600tccaggaata tggcaattag attgtacaca tctggaagga aaagttatcc tggtagcagt 3660tcatgtagcc agtggctata tagaagcaga agttattcca gcagaaacgg ggcaagaaac 3720agcatacttt ctcttaaaat tagcaggaag atggccagta aaagtagtac atacagataa 3780tggcagcaat ttcaccagtg ctacagttaa ggccgcctgt tggtgggcag ggatcaaaca 3840ggaatttgga attccctaca atccccaaag tcaaggagta gtagaatcta tgaataaaga 3900attaaagaaa attataggac aggtaagaga tcaagctgaa catcttaaga cagcagtaca 3960aatggcagta tttatccaca attttaaaag aaaagggggg attgggggat acagtgcagg 4020ggaaagaata atagacataa tagcaacaga catacaaact agagaattac aaaaacaaat 4080cataaaaatt caaaattttc gggtttatta cagggacagc agagatccaa tttggaaagg 4140accagcaaag cttctctgga aaggtgaagg ggcagtagta atacaagaca atagtgacat 4200aaaggtagta ccaagaagaa aagtaaagat cattagggat tatggaaaac agatggcagg 4260tgatgattgt gtggcaagta gacaggatga ggattaac 429821499PRTHuman immunodeficiency virus type 1 2Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Ile Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Ile Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn 65 70 75 80 Thr Ile Ala Thr Leu Tyr Cys Val His Glu Arg Ile Glu Val Lys Asp 85 90 95 Thr Lys Glu Ala Val Glu Lys Met Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110 Lys Lys Thr Gln Gln Ala Ala Ala Asp Ser Ser Gln Val Ser Gln Asn 115 120 125 Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 130 135 140 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys Ala 145 150 155 160 Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 165 170 175 Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln 180 185 190 Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Asp Glu Ala Ala Glu 195 200 205 Trp Asp Arg Leu His Pro Val His Ala Gly Pro Val Ala Pro Gly Gln 210 215 220 Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu 225 230 235 240 Gln Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly 245 250 255 Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 260 265 270 Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 275 280 285 Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu 290 295 300 Gln Ala Ser Gln Asp Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 305 310 315 320 Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 325 330 335 Gln Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 340 345 350 Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr 355 360 365 Gly Ser Val Thr Ala Val Met Met Gln Arg Gly Asn Phe Lys Gly Pro 370 375 380 Arg Lys Ser Ile Lys Cys Phe Asn Cys Gly Lys Glu Gly His Thr Ala 385 390 395 400 Lys Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Arg 405 410 415 Glu Gly His Gln Met Lys Asp Cys Ser Glu Arg Gln Ala Asn Phe Leu 420 425 430 Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe Leu Gln 435 440 445 Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser Phe Gly Phe Gly 450 455 460 Glu Glu Ile Thr Pro Ser Gln Lys Gln Glu Gln Lys Asp Lys Glu Leu 465 470 475 480 Tyr Pro Leu Ala Ser Leu Lys Ser Leu Phe Gly Asn Asp Pro Ser Ser 485 490 495 Gln Phe Phe Arg Glu Asp Leu Ala Phe Pro Gln Gly Lys Ala Gly Glu 500 505 510 Phe Ser Ser Glu Gln Thr Arg Ala Asn Ser Pro Thr Ser Arg Glu Leu 515 520 525 Arg Val Trp Gly Gly Asp Asn Pro Leu Ser Glu Thr Gly Ala Glu Gly 530 535 540 Gln Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg 545 550 555 560 Pro Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu 565 570 575 Asp Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly 580 585 590 Lys Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val 595 600 605 Arg Gln Tyr Asp Gln Ile Leu Ile Glu Ile Cys Gly Tyr Lys Ala Met 610 615 620 Gly Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn 625 630 635 640 Leu Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile 645 650 655 Glu Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val 660 665 670 Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Thr Glu Ile 675 680 685 Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Arg Ile Gly Pro Glu 690 695 700 Asn Pro Tyr Asn Thr Pro Ile Phe Ala Ile Lys Lys Lys Asp Ser Thr 705 710 715 720 Lys Trp Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln 725 730 735 Asp Phe Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys 740 745 750 Lys Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 755 760 765 Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro 770 775 780 Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu 785 790 795 800 Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr 805 810 815 Lys Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Glu Ile Val Ile Tyr 820 825 830 Gln Tyr Met Asp Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln 835 840 845 His Arg Thr Lys Ile Glu Glu Leu Arg Glu His Leu Leu Arg Trp Gly 850 855 860 Phe Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp 865 870 875 880 Met Gly Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Lys 885 890 895 Leu Pro Glu Lys Glu Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val 900 905 910 Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Lys 915 920 925 Gln Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Val 930 935 940 Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile 945 950 955 960 Leu Lys Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu 965 970 975 Ile Ala Glu Leu Gln Lys Gln Gly Asp Gly Gln Trp Thr Tyr Gln Ile 980 985 990 Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Thr 995 1000 1005 Arg Gly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val 1010 1015 1020 Gln Lys Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro 1025 1030 1035 Lys Phe Lys Leu Pro Ile Gln Lys Glu Thr Trp Glu Thr Trp Trp 1040 1045 1050 Ile Glu Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe Val 1055 1060 1065 Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu 1070 1075 1080 Pro Ile Ile Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn 1085 1090 1095 Arg Glu Thr Lys Leu Gly Lys Ala Gly Tyr Val Thr Asp Arg Gly 1100 1105 1110 Arg Gln Lys Val Val Pro Phe Thr Asp Thr Thr Asn Gln Lys Thr 1115 1120 1125 Glu Leu Gln Ala Ile Asn Leu Ala Leu Gln Asp Ser Gly Leu Glu 1130 1135 1140 Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile Gln 1145 1150 1155 Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Ser Gln Ile Ile 1160 1165 1170 Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu Ala Trp Val Pro 1175 1180 1185 Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val 1190 1195 1200 Ser Gln Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys 1205 1210 1215 Ala Gln Glu Glu His Glu Lys Tyr His Asn Asn Trp Arg Ala Met 1220 1225 1230 Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val 1235 1240 1245 Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly 1250 1255 1260 Gln Val Asp Cys Ser Pro Gly Ile Trp Gln Leu Asp Cys Thr His 1265 1270 1275 Leu Glu Gly Lys Val Ile Leu Val Ala Val His Val Ala Ser Gly 1280 1285 1290 Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr 1295 1300 1305 Ala Tyr Phe Leu Leu Lys Leu Ala Gly Arg Trp Pro Val Lys Val 1310 1315 1320 Val His Thr Asp Asn Gly Ser Asn Phe Thr Ser Ala Thr Val Lys 1325 1330 1335 Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu Phe Gly Ile Pro 1340 1345 1350 Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met Asn Lys Glu 1355 1360 1365 Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu 1370 1375 1380 Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys Arg 1385 1390 1395 Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp 1400 1405 1410 Ile Ile Ala Thr Asp Ile Gln Thr Arg Glu Leu Gln Lys Gln Ile 1415 1420 1425 Ile Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp 1430 1435 1440 Pro Ile Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly 1445 1450 1455 Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg 1460 1465 1470 Arg Lys Val Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly 1475 1480 1485 Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp 1490 1495 3129PRTHuman immunodeficiency virus type 1 3Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Thr Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Ile Gln Thr Gly Ser Glu Glu Ile Arg Ser Leu Tyr Asn 65 70 75 80 Thr Val Ala Thr Leu Tyr Cys Val His Glu Arg Ile Glu Val Lys Asp 85 90 95 Thr Lys Glu Ala Val Glu Lys Met Glu Glu Glu

Gln Asn Lys Ser Lys 100 105 110 Lys Lys Thr Gln Gln Ala Ala Ala Asp Ser Ser Gln Val Ser Gln Asn 115 120 125 Tyr 4129PRTHuman immunodeficiency virus type 1 4Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Thr Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Ile Gln Thr Gly Ser Glu Glu Ile Arg Ser Leu Tyr Asn 65 70 75 80 Thr Val Ala Thr Leu Tyr Cys Val His Glu Arg Ile Glu Val Lys Asp 85 90 95 Thr Lys Glu Ala Val Glu Lys Met Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110 Lys Lys Thr Gln Gln Ala Ala Ala Asp Ser Ser Gln Val Ser Gln Asn 115 120 125 Tyr 5132PRTHuman immunodeficiency virus type 1 5Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn 65 70 75 80 Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Ile Lys Asp 85 90 95 Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110 Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly His Ser Asn Gln Val 115 120 125 Ser Gln Asn Tyr 130 675PRTHuman immunodeficiency virus type 1 6Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 773PRTHuman immunodeficiency virus type 1 7Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu 35 40 45 Leu Glu Thr Thr Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu Gln Pro 50 55 60 Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 875PRTHuman immunodeficiency virus type 1 8Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Gln Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Glu Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 975PRTHuman immunodeficiency virus type 1 9Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Gly Gly Cys Arg Gln Ile Leu Glu Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 1075PRTHuman immunodeficiency virus type 1 10Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Ile Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Ala Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 His Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 1173PRTHuman immunodeficiency virus type 1 11Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Lys Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Ala Gln Leu Gln Pro 50 55 60 Ser Leu Pro Thr Gly Ser Glu Glu Leu 65 70 1275PRTHuman immunodeficiency virus type 1 12Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 1373PRTHuman immunodeficiency virus type 1 13Ala Arg Ala Ser Ile Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Ile Gln Ile Leu Gly Gln Leu Gln Pro 50 55 60 Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 1475PRTHuman immunodeficiency virus type 1 14Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Val Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Lys Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 1575PRTHuman immunodeficiency virus type 1 15Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Gln Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Glu Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Ile 65 70 75 1675PRTHuman immunodeficiency virus type 1 16Met Gly Ala Arg Ala Ser Ile Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Gln Tyr Arg Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Gly Gly Cys Lys Gln Ile Leu Ala Gln Leu 50 55 60 His Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 1773PRTHuman immunodeficiency virus type 1 17Ala Arg Ala Ser Ile Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Arg Tyr Arg Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Arg Gln Leu Gln Pro 50 55 60 Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 1875PRTHuman immunodeficiency virus type 1 18Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Arg Gly Lys Lys Lys Tyr Gln Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ser Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Arg Gln Leu 50 55 60 Gln Pro Ala Leu Gln Thr Gly Ser Glu Asp Phe 65 70 75 1975PRTHuman immunodeficiency virus type 1 19Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2075PRTHuman immunodeficiency virus type 1 20Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Ser Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Ser Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2175PRTHuman immunodeficiency virus type 1 21Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Gln Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Ser Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2275PRTHuman immunodeficiency virus type 1 22Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Lys Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2375PRTHuman immunodeficiency virus type 1 23Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Val Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Lys Gln Ile Leu Ala Gln Leu 50 55 60 His Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2472PRTHuman immunodeficiency virus type 1misc_feature(18)..(18)Xaa can be any naturally occurring amino acidmisc_feature(51)..(51)Xaa can be any naturally occurring amino acid 24Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp Glu Lys Ile 1 5 10 15 Arg Xaa Arg Gln Gly Gly Lys Lys Lys Tyr Lys Leu Lys His Ile Val 20 25 30 Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro Gly Leu Leu 35 40 45 Glu Thr Xaa Glu Gly Cys Arg Gln Ile Leu Glu Gln Leu Gln Pro Ala 50 55 60 Leu Gln Thr Gly Ser Glu Glu Leu 65 70 2575PRTHuman immunodeficiency virus type 1 25Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Gly Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2675PRTHuman immunodeficiency virus type 1 26Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Thr Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Ile Gln Thr Gly Ser Glu Glu Ile 65 70 75 2775PRTHuman immunodeficiency virus type 1 27Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 65 70 75 2875PRTHuman immunodeficiency virus type 1 28Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Thr Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Gly Gln Leu 50 55 60 Gln Pro Ser Ile Gln Thr Gly Ser Glu Glu Ile 65 70 75 2975PRTHuman immunodeficiency virus type 1 29Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Ile Ser Gln Leu 50 55 60 Gln Pro Ser Leu Lys Thr Gly Ser Glu Glu Leu 65 70 75 3075PRTHuman immunodeficiency virus type 1 30Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Glu Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Thr Tyr Lys Leu Lys 20 25

30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Ala Gln Leu 50 55 60 Gln Ser Ser Ile Gln Thr Gly Ser Glu Glu Ile 65 70 75 3173PRTHuman immunodeficiency virus type 1 31Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Glu Trp Glu Lys 1 5 10 15 Ile Gln Leu Arg Pro Gly Gly His Lys Arg Tyr Lys Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Ile Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Gly Gly Cys Arg Gln Ile Met Gly Gln Leu Gln Pro 50 55 60 Ala Ile Gln Thr Gly Ser Glu Glu Leu 65 70 3273PRTHuman immunodeficiency virus type 1 32Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Arg Lys Arg Tyr Arg Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile Ser Gln Leu Gln Pro 50 55 60 Ser Leu Lys Thr Gly Ser Glu Glu Leu 65 70 3361PRTHuman immunodeficiency virus type 1 33Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu 50 55 60 3461PRTHuman immunodeficiency virus type 1 34Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Ala Leu Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Thr Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile 50 55 60 3561PRTHuman immunodeficiency virus type 1 35Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Ile 50 55 60 3661PRTHuman immunodeficiency virus type 1 36Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Glu Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Thr Tyr Lys Leu Lys 20 25 30 His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile 50 55 60 3759PRTHuman immunodeficiency virus type 1 37Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Glu Trp Glu Lys 1 5 10 15 Ile Gln Leu Arg Pro Gly Gly His Lys Arg Tyr Lys Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Ile Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Gly Gly Cys Arg Gln Ile Met 50 55 3859PRTHuman immunodeficiency virus type 1 38Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Arg Lys Arg Tyr Arg Leu Lys His Ile 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Lys Gln Ile Ile 50 55 3961PRTHuman immunodeficiency virus type 1 39Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Arg Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Lys Gln Leu Met 50 55 60 4061PRTHuman immunodeficiency virus type 1misc_feature(54)..(54)Xaa can be any naturally occurring amino acidmisc_feature(61)..(61)Xaa can be any naturally occurring amino acid 40Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ser Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Met Glu Arg Phe Ala Leu Asn Pro 35 40 45 Asp Leu Leu Glu Thr Xaa Glu Gly Cys Gln Gln Ile Xaa 50 55 60 4159PRTHuman immunodeficiency virus type 1 41Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Ile Lys His Leu 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Met 50 55 4261PRTHuman immunodeficiency virus type 1 42Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Arg Tyr Arg Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Asp Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Lys Ile Met 50 55 60 4361PRTHuman immunodeficiency virus type 1 43Met Gly Ala Arg Ala Ser Val Leu Thr Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Ser Tyr Lys Ile Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Asp Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Met 50 55 60 4461PRTHuman immunodeficiency virus type 1 44Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Asp Arg Phe Ala Leu Asn Pro 35 40 45 Ser Leu Leu Glu Thr Ser Glu Gly Cys Gln Gln Ile Ile 50 55 60 4561PRTHuman immunodeficiency virus type 1 45Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Asp Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Leu 50 55 60 4661PRTHuman immunodeficiency virus type 1misc_feature(42)..(42)Xaa can be any naturally occurring amino acidmisc_feature(49)..(49)Xaa can be any naturally occurring amino acid 46Met Gly Ala Arg Ala Ser Ile Leu Ser Gly Gly Arg Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Xaa Arg Phe Ala Leu Asn Pro 35 40 45 Xaa Leu Leu Glu Ser Ala Glu Gly Cys Gln Gln Ile Met 50 55 60 4759PRTHuman immunodeficiency virus type 1 47Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp Glu Lys 1 5 10 15 Ile Gln Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Leu 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro Asp Leu 35 40 45 Leu Glu Thr Ser Glu Gly Cys Gln Gln Ile Ile 50 55 4859PRTHuman immunodeficiency virus type 1 48Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ser Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Leu 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro Ser Leu 35 40 45 Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Met 50 55 4961PRTHuman immunodeficiency virus type 1 49Met Gly Ala Arg Ala Ser Val Leu Arg Gly Glu Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Met Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Ala Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Arg Gln Ile Ile 50 55 60 5061PRTHuman immunodeficiency virus type 1 50Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Gln Leu Arg Pro Gly Gly Lys Lys Arg Tyr Met Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Arg Gln Ile Ile 50 55 60 5161PRTHuman immunodeficiency virus type 1 51Met Gly Ala Arg Ala Ser Val Leu Arg Gly Glu Lys Leu Asp Thr Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Met Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Ala Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Arg Gln Ile Ile 50 55 60 5261PRTHuman immunodeficiency virus type 1 52Met Gly Ala Arg Ala Ser Ile Leu Arg Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Gln Leu Arg Pro Gly Gly Lys Lys Arg Tyr Met Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Arg Gln Ile Ile 50 55 60 5359PRTHuman immunodeficiency virus type 1 53Ala Arg Ala Ser Ile Leu Arg Gly Gly Gln Leu Asp Arg Trp Glu Lys 1 5 10 15 Ile Arg Leu Arg Pro Gly Gly Lys Lys His Tyr Met Leu Lys His Leu 20 25 30 Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Val Leu Asn Pro Gly Leu 35 40 45 Leu Glu Thr Ala Glu Gly Cys Lys Gln Ile Met 50 55 5461PRTHuman immunodeficiency virus type 1 54Met Gly Ala Arg Ala Ser Ile Leu Thr Gly Glu Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Met Ile Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile 50 55 60 5561PRTHuman immunodeficiency virus type 1 55Met Gly Ala Arg Ala Ser Ile Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Leu Ile 50 55 60 5661PRTHuman immunodeficiency virus type 1 56Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Gln Leu Lys 20 25 30 His Val Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile 50 55 60 5761PRTHuman immunodeficiency virus type 1 57Met Gly Ala Arg Ala Ser Ile Leu Thr Gly Glu Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Met Ile Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile 50 55 60 5861PRTHuman immunodeficiency virus type 1 58Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Asp Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Gln Tyr Lys Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Lys Ile Ile 50 55 60 5961PRTHuman immunodeficiency virus type 1 59Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Ile Trp Ala Gly Arg Glu Leu Asp Arg Phe Ala Leu Asp Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Lys Ile Ile 50 55 60 6061PRTHuman immunodeficiency virus type 1 60Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asp Ser 35 40 45 Gly Leu Leu Glu Thr Thr Glu Gly Cys Arg Lys Ile Ile 50 55 60 6161PRTHuman immunodeficiency virus type 1 61Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Lys Tyr Lys Met Lys 20 25 30 His Leu Ile Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asp Pro 35 40 45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Lys Ile Ile 50 55 60 6238PRTHuman immunodeficiency virus type 1 62Gly Gly Lys Lys Lys Tyr Arg Leu Lys His Leu Val Trp Ala Ser Arg 1 5 10 15 Glu Leu Glu Arg Phe Ala Leu Asn Pro Gly Leu Leu Glu Thr Thr Glu 20 25 30 Gly Cys Lys Gln Ile Ile 35 6361PRTHuman immunodeficiency virus type 1 63Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Arg Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Asp Leu Leu Glu Thr Ala Asp Gly Cys Gln Gln Ile Leu 50 55 60 6461PRTHuman immunodeficiency virus type 1

64Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Asp Leu Leu Asp Thr Ala Glu Gly Cys Leu Gln Leu Ile 50 55 60 6561PRTHuman immunodeficiency virus type 1 65Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Thr Pro Glu Gly Cys Leu Gln Ile Ile 50 55 60 6661PRTHuman immunodeficiency virus type 1misc_feature(61)..(61)Xaa can be any naturally occurring amino acid 66Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Leu Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Asp Arg Phe Ala Leu Asn Pro 35 40 45 Asp Leu Leu Glu Thr Ala Asp Gly Cys Leu Lys Ile Xaa 50 55 60 6761PRTHuman immunodeficiency virus type 1 67Met Gly Ala Arg Ala Ser Ile Leu Ser Gly Gly Lys Leu Asp Asp Trp 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Gln Tyr Arg Ile Lys 20 25 30 His Leu Val Trp Ala Ser Arg Glu Leu Asp Arg Phe Ala Leu Asn Pro 35 40 45 Gly Leu Leu Glu Ser Ala Lys Gly Cys Gln Gln Ile Leu 50 55 60

Claims

1. A replication-defective lentiviral packaging vector lacking a 4) site and encoding an HIV-1 Gag MA protein; wherein the HIV-1 Gag MA protein: a) has an amino acid at position 12 that is not a glutamic acid, b) has an amino acid at position 15 that is not an arginine, c) does not have both a valine at position 46 and a leucine at position 61; and wherein the lentiviral packaging vector encoding the HIV-1 Gag MA protein generates at least a 1.5 fold increase in the titer of a packaged lentiviral vector as compared to the lentiviral packaging vector encoding an HIV-1 Gag MA protein of HIV-1 BRU.

2. The vector of claim 1, wherein the amino acid at position 12 is a lysine.

3. The vector of claim 1, wherein the amino acid at position 12 is an arginine.

4. The vector of claim 1, wherein the amino acid at position 15 is a threonine.

5. The vector of claim 1, wherein the amino acid at position 15 is an alanine.

6. The vector of claim 1, wherein the amino acid at position 46 is a leucine.

7. The vector of claim 1, wherein the amino acid at position 61 is an isoleucine.

8. The vector of claim 1, wherein the amino acid at position 61 is a methionine.

9. The vector of claim 2, wherein the amino acid at position 15 is a threonine.

11. The vector of claim 2, wherein the amino acid at position 46 is a leucine.

12. The vector of claim 2, wherein the amino acid at position 61 is an isoleucine.

13. The vector of claim 9, wherein the amino acid at position 46 is a leucine.

14. The vector of claim 9, wherein the amino acid at position 61 is an isoleucine.

15. The vector of claim 13, wherein the amino acid at position 61 is an isoleucine.

16. The vector of claim 15, wherein the HIV-1 Gag MA protein is the HIV-1 Gag MA protein of HIV-1 NDK.

17. The vector of claim 16, wherein the vector encodes the amino acid sequence of SEQ ID NO:4.

18. A method for generating a packaging vector comprising inserting a Gag and/or Pol sequence from a non-subtype B HIV-1 virus into a plasmid under the control of a non-HIV promoter to generate the packaging vector.

19. A method for generating a lentiviral vector comprising administering a packaging vector encoding a non-subtype B HIV-1 Gag or Pol protein to a cell with a lentiviral vector.

20. The method of claim 19, further comprising selecting for a packaging vector that packages a higher titer of the lentiviral vector than a same packaging vector encoding a subtype B HIV-1 Gag or Pol protein.