METHOD OF INDUCING THE PRODUCTION OF PROTECTIVE ANTI-HIV-1 ANTIBODIES

Abstract

The present invention relates, in general, to an immunogen for HIV vaccination and, in particular, to a method of inducing the production of protective anti-HIV antibodies by targeting B cell germline and clone intermediates using a combination of HIV envelope and non-HIV immunogens. The invention also relates to compositions suitable for use in such a method.

Description

[0001] This application is a Continuation of U.S. Ser. No. 13/581,157, filed on Aug. 24, 2012, which is the U.S. national phase of International Application No. PCT/US2011/000352, filed on Feb. 25, 2011, which designated the U.S. and claims priority to U.S. Provisional Application No. 61/282,526, filed Feb. 25, 2010, U.S. Provisional Application No. 61/344,457, filed Jul. 27, 2010, U.S. Provisional Application No. 61/344,580, filed Aug. 25, 2010 and U.S. Provisional Application No. 61/344,622, filed Sep. 1, 2010, the entire contents of each which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 12, 2016, is named 02933311-035US7_SL.txt and is 108,945 bytes in size.

TECHNICAL FIELD

[0004] The present invention relates, in general, to an immunogen for HIV-1 vaccination and, in particular, to a method of inducing the production of protective anti-HIV-1 antibodies by targeting B cell germline and clone intermediates using a combination of non-HIV-1 and HIV-1 immunogens. The invention also relates to compositions suitable for use in such a method.

BACKGROUND

[0005] The first antibody response to transmitted/founder HIV-1 envelope is non-neutralizing, targets Env gp41 and occurs at a mean of 13 days after appearance of plasma viremia (Tomaras et al, J. Virology 82:12449-63 (2008)). While the initial T cell response to HIV-1 that occurs at the same time as the initial antibody response drives mutations within T cell epitopes of HIV-1, the initial gp41 antibody response to HIV-1 does not. Rather, it is the autologous neutralizing antibody response, which is delayed until approximately three months after transmission, that is the first neutralizing antibody response associated with antibody escape mutants (McMichael et al, Nature Rev. Immunol. 10:11-23 (2010)).

[0006] The four epitopes on HIV-1 envelope to which rare broadly reactive neutralizing antibodies bind are the CD4 binding site (CD4BS) (mab (monoclonal antibody) IgG1b12) (Zwick et al, J. Virol. 77(10):5863-5876 (2003)), the membrane proximal external region (MPER) epitopes defined by human mabs 2F5 and 4E10 (Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004), Stiegler and Katinger, J. Antimicrob. Chemother. 51:757-759 (2003), Zwick et al, Journal of Virology 79:1252-1261 (2005), Purtscher et al, AIDS 10:587 (1996)), and the mannan glycan epitope defined by human mab 2G12 (Scanlan et al, Adv. Exper. Med. Biol. 535:205-218 (2003)). These four rare human mabs are all unusual: two are IgG3 (2F5 and 4E10), one has a unique Ig dimer structure (2G12), one has a very hydrophobic CDR3 (2F5) (Ofek et al, J. Virol. 198:10724 (2004)), and, in all four, the CDR3 is unusually long (Burton et al, Nature Immunol. 5(3):233-236 (2004), Kunert et al, AIDS Res. Hum. Retroviruses 20(7):755-762 (2004), Zwick et al, J. Virol. 78(6):3155-3161 (2004), Cardoso et al, Immunity 22:163-172 (2005)). Of these, 2F5- and 4E10-like human mabs are quite rare. Acute HIV-1 patients do not make antibodies against the MPER or 2G12 epitopes, MPER can be defined as amino acids 652 to 683 of HIV envelope (Cardoso et al, Immunity 22:163-173 (2005) (e.g., QQEKNEQELLELDKWASLWNWFDITNWLWYIK) (SEQ ID NO: 1). CD4 binding site (BS) antibodies are commonly made early in HIV-1 infection, but these antibodies generally do not have the broad spectrum of neutralization shown by mab IgG1b12 (Burton et al, Nat. Immunol. 5(3):233-236 (2004)).

[0007] To understand the pathogenesis of the ineffective initial antibody response to HIV-1 envelope (Env), PCR has been performed for amplification of immunoglobulin variable region of heavy- and light-chain (V.sub.H and V.sub.L) genes from single blood or bone marrow plasma cells from 5 acutely infected subjects from 17-30 days after HIV-1 transmission. The specificities of the plasma cell response induced by HIV-1 infection have been determined. Using PCR amplification of V.sub.H and V.sub.L genes of single human plasma cells induced by transmitted HIV-1, the initial plasma cell/plasmablast response to HIV-1 has been studied. It has been found that the first antibody response to HIV-1 is induced to HIV-1 Env gp41, and that gp41 induces an antibody response in pre-existing memory B cell clones, resulting in low-affinity, polyreactive anti-Env antibodies that cross-react with a number of host and bacterial molecules, particularly, of human gut bacterial flora.

[0008] The present invention results, at least in part, from studies designed to identify the source of both the initial anti-HIV-1 Env gp41 antibodies and the rare broadly neutralizing antibodies. The invention further results from the identification of a cellular protein expressed in most warm blooded vertebrates that is structurally similar to the 2F5, and possibly 4E10, epitopes of the HIV-1 gp41 MPER.

[0009] The invention provides an HIV-1 vaccine designed to target a naive B cell pool that can be driven to give rise to broadly neutralizing antibodies to HIV-1.

SUMMARY OF THE INVENTION

[0010] In general, the present invention relates to an immunogen for HIV vaccination. More specifically, the invention relates to a method of inducing the production of protective anti-HIV-1 antibodies by targeting B cell germline and clone intermediates using a combination of non-HIV-1 and HIV-1 immunogens. The invention also relates to compositions suitable for use in such a method.

[0011] Objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1. A representative influenza antibody clone against H1 Soloman Islands hemagglutinin.

[0013] FIG. 2. Plasma cell antibody repertoire in patient 684-6, .about.20 days after HIV-1 transmission.

[0014] FIG. 3. Production of inferred intermediate clone antibodies.

[0015] FIG. 4. Inferred germline and clone member intermediates assayed for reactivity with clade B gp41, autologous gp140 and group M consensus gp140 to determine where in the clone development reactivity with gp41 was acquired.

[0016] FIG. 5. Reactivity of clone 684-6B acquired at the second intermediate precursor antibody (see also FIG. 4).

[0017] FIG. 6. Additional inferred intermediate antibody clones produced in mg quantities and analyzed for the dissociation constants (Kd) of antibody binding to gp41.

[0018] FIG. 7. Acquisition of gp41 reactivity in patient 684-6 clone 684-6B germline and inferred intermediate antibodies.

[0019] FIG. 8. Polyreactivity of 6846 clone 52 germline and inferred intermediate gp41 antibodies.

[0020] FIG. 9. Reactivity of aerobic gut flora with clone 684-6B antibodies.

[0021] FIGS. 10A and 10B. Blue Native-PAGE and western blot images of gut extract vs Mojo antibody. FIG. 10A. Coomassie blue image. FIG. 10B. Western blot image.

[0022] FIG. 11. Western blot image of gut extract vs Mojo antibody--non-reducing vs HV00276.

[0023] FIG. 12. Western blot image of gut extract vs Mojo antibody--reducing vs HV00276.

[0024] FIGS. 13A and 13B. 1b12 germline antibody binds to lipids (PC:CL liposomes). FIG. 13A. Binding to HIV 89.6 gp120. FIG. 13B. Binding to lipids (PC:cardiolipin).

[0025] FIG. 14. A large fraction of B cells expressing 4E10 V.sub.H are deleted in bone marrow at the pre-B to immature B cell stage in 4E10 V.sub.H knock-in mice.

[0026] FIG. 15. Two roadblocks for induction of broad neutralizing antibodies. The first roadblock is that vaccines currently designed to stimulate B cells that produce rare broad neutralizing antibodies do not react with the germline B cell receptors of the naive B cells that are required to respond to the immunogen. While the initial B cell response to HIV-1 Env is made early on after infection, there is a cross reactivity of gp41 with host or pre-existing foreign molecules such that the B cell antibody clones that make the initial gp41 antibody response are derived from pre-existing polyreactive natural B cell clones whose germlines also do not react with gp41 and whose reactivity to gp41 is acquired later in clonal antibody development as cross-reactivity with gp41 is acquired through host or foreign antigen-driven clonal expansion. Once gp41 reactivity is acquired, gp41 then drives the clonal expansion. The second roadblock to vaccine development comes from work showing that both of these antibodies require the long hydrophobic CDR3s with lipid reactivity to neutralize (Alam et al, Proc. Natl. Acad. Sci. USA 106:20234-9 (2009)) and that the 2F5 and 4E10 V.sub.HS are sufficiently autoreactive to promote deletion in knock-in mice (Verkoczy et al, Proc. Natl. Acad. Sci. USA 107:181-186 (2010)).

[0027] FIGS. 16A-16F. Strategy for induction of broad neutralizing antibodies. FIG. 16A. Vaccines must be designed to stimulate B cell precursors by inclusion of either host (such as lipids) and/or foreign (such as gut flora) antigens to which the polyreactive naive B cell receptors (BCRs) bind (left-most arrow), and antigens (preferred Env constructs) to target intermediate clones of B cells that arise that cross-react with Env. The Env lead candidates for this component of the vaccine is the Malawi 1086 clade C gp140 oligomer that has induced in guinea pigs considerable breadth in neutralizing antibodies mixed with the clade B JRFL gp140 Env that selectively expresses the MPER neutralizing epitopes (middle arrow) and/or the transmitted founder Envs 6240, 040 and 63521 (see FIGS. 16B, 16C and 16D) that preferentially express epitopes bound by broadly neutralizing monoclonal antibodies. Finally, to overcome peripheral deletion and/or anergy of B cells that are driven to make polyreactive neutralizing antibodies, the vaccine contains potent TLR agonists or other adjuvants to drive activation of polyreactive B cells by germline and intermediate clone-targeted vaccines (right-most arrow). FIG. 16E. SDS-PAGE images of apoferritin. FIG. 16F. Western blot images of apoferritin vs HV00274, HV00276. Acute HIV infection gp41 inferred intermediate antibodies 276 from clone 684-6B and 274 from clone 684-6A both bind to the 19Kd apoferritin subunit. Both mabs also bind to the 60Kd protein in the native marker.

[0028] FIG. 17. Design of HIV-1 Env gp140 constructs.

[0029] FIG. 18. Analysis of acute HIV-1 Envs and Group M consensus HIV-1 Env by Blue Native-PAGE and SDS-PAGE.

[0030] FIGS. 19A and 19B. FIG. 19A. Immunogenicity of Group M Consensus HIV-1 Env, CON-S and Subtype C Acute HIV-1 Env, 1086C, Subtype B chronic HIV-1 Env, JRFL. FIG. 19B. Methods. FIG. 19B discloses SEQ ID NOS 9-10, respectively, in order of appearance.

[0031] FIG. 20. Deglycosylation of JRFL Env gp 140 CF with PNGase.

[0032] FIGS. 21A and 21B. Antigenicity of JRFL HIV Env gp140CF in ELISA.

[0033] FIG. 22. Antigenicity of JRFL gp140 Env in SPR.

[0034] FIG. 23. Fusion-intermediate state of HIV-1 gp41 targeted by broadly neutralizing antibodies. FIG. 23 discloses "His6" as SEQ ID NO: 11.

[0035] FIGS. 24A and 24B. FIG. 24A. Design of membrane anchored gp41-inter. FIG. 24B. 2F5 and 4E10 mAbs bind to membrane conjugated gp-41-inter with nM Kd and almost irreversible off-rates. FIG. 24A discloses "His6" as SEQ ID NO: 11.

[0036] FIG. 25. Lead candidate immunogens.

[0037] FIG. 26. Gp41-inter liposomes with TLR ligands and encapsulated immunomodulatory ligands.

[0038] FIG. 27. Amino acid sequences for HIV-1 transmitted founder Envs 1086.C, 089.C, 040_C9, and 63521, and codon optimized encoding sequences. FIG. 27 discloses SEQ ID NOS 12-19, respectively, in order of appearance.

[0039] FIG. 28. Clade B JRFL and 6240 gp140 Env sequence and encoding sequence. FIG. 28 discloses SEQ ID NOS 20-23, respectively, in order of appearance.

[0040] FIG. 29. Early B cell response to HIV-1: the role of innate B cells.

[0041] FIG. 30. 2F5 and 4E10 broadly neutralizing antibodies react with self antigens that are phylogenetically conserved

[0042] FIG. 31. 2F5 specifically binds to 43 kDa, 50 kDa, 70 kDa and 350 kDa 3T3 (mouse) cellular proteins on western blot

[0043] FIG. 32. Conserved self-antigens that carry the 2F5 nominal epitope. FIG. 32 discloses SEQ ID NOS 2, 24 and 24-25, respectively, in order of appearance.

[0044] FIG. 33. The H3 domain of kynuereninase (KYNU) is highly conserved. FIG. 33 discloses SEQ ID NOS 26-36, respectively, in order of appearance.

[0045] FIG. 34. Structure of human KYNU (PDB 2HZP) and location of ELDKWA (SEQ ID NO:2) motif.

[0046] FIG. 35. Illustration of the DKW residues (ELDKWA) (SEQ ID NO: 2) in human KYNU.

[0047] FIG. 36. Binding of the 2F5 antibody to human KYNU may require distortion of the H3 domain. FIG. 36 discloses "ELDKWA" as SEQ ID NO: 2.

[0048] FIG. 37. KYNU dimers likely obscure the potential 2F5 binding site. FIG. 37 discloses "ELDKWA" as SEQ ID NO: 2.

[0049] FIG. 38. 2F5 and possibly 4E10 antibodies bind to recombinant human KYNU in western blots.

[0050] FIG. 39. KYNU is recognized by 2F5-family antibodies.

[0051] FIG. 40. 2F5 antibody avidly reacts with rhKYNU in a standard ELISA.

[0052] FIG. 41. 2F5 antibody reacts with a peptide (DP178-Q16L) containing 2F5 epitope--anti-KYNU antibody does not.

[0053] FIG. 42. 2F5 binding to rhKYNU and DP178-Q16L is comparable in a standard ELISA.

[0054] FIG. 43. Antibody binding in ELISA plates is antigen specific.

[0055] FIG. 44. 13H11 does not bind rhKYNU.

[0056] FIG. 45. 13H11 reacts with DP178-Q16L but not MPER-656. FIG. 45 discloses SEQ ID NOS 24 and 37-38, respectively, in order of appearance.

[0057] FIG. 46. Competitive inhibition of 2F5 binding to rhKYNU by JRFL, DP178-Q16L and R4A.

[0058] FIG. 47. Comparable inhibition of 2F5 binding to rhKYNU and JRFL.

[0059] FIG. 48. Soluble KYNU is bound by 2F5.

[0060] FIG. 49. rhKYNU binding to surface-captured mAbs.

[0061] FIGS. 50A-50C. Binding of 2F5 mAb and 2F5 RUA (reverted unmutated ancestor) antibodies to KYNU, (FIG. 50A) 2F5, (FIG. 50B) 2F5-GL1, (FIG. 50C) 2F5-GL3.

[0062] FIG. 51. Inhibition of 2F5 binding to 3T3 cells by recombinant HIV-1 gp140 (JRFL), and the DP178 and R4A peptides.

[0063] FIGS. 52A-52D. Enrichment and identification of protein band in intestinal bacterial lysate reactive with mAb HV00276. (FIG. 52A) Western blot analysis following Native PAGE gel run. (FIG. 52B) Protein fractions from bacterial lysate with molecular wt .about.500 kDa collected following size exclusion chromatography (SEC). (FIG. 52C) SEC fractions show enrichment of 520 kDa protein by Coomassie Blue (1) and silver staining (2) and western blotting (3, arrow). (FIG. 52D) Isoelectric zoom fractionation.

[0064] FIGS. 53A-53C. Liquid chromatography-mass spectrometry (LC-MS) identification of RNA polymerase. (FIG. 53A) LC-MS identification of RNA polymerase .beta. subunit (SEQ ID NO: 39). (FIG. 53B) LC-MS identification of RNA polymerase .beta.' subunit (SEQ ID NO: 40). (FIG. 53C) LC-MS identification of RNA polymerase .alpha. subunit (SEQ ID NO: 41).

[0065] FIG. 54. Mab HV00276 binds to RNA polymerase core protein.

[0066] FIG. 55. Mab HV00276 binds to the .alpha. subunit of RNA polymerase core protein.

[0067] FIG. 56. Neutralization screening of primary memory B cell cultures. Memory B cells from peripheral blood of CHAVI08 chronically-HIV-1 infected volunteer 707-01-021-9 were EBV-transformed and stimulated for 14 days in presence of CD40 ligand, oCpGs and CHK-2 inhibitor at a density of 8 cells/well. At the end of stimulation supernatants were tested for neutralizing activity against the reporter tier 2 clade C CAP45 virus. Solid dots represent the percentage of neutralization of each of the 3,600 cultures. Monoclonal antibodies CH01-CH05 were isolated from the cultures represented with open dotted symbols. Positive controls (HIV Ig) are shown as open circles on the far right.

[0068] FIGS. 57A-57C. V-heavy and V-light chain alignments of monoclonal antibodies CH01-CH05. Alignment of the sequences of the CH01-CH05 V-heavy chains (SEQ ID NOS 42-47, respectively, in order of appearance) (FIG. 57A), CH01-CH04 (SEQ ID NOS 48-52, respectively, in order of appearance) (FIG. 57B) and CH05 (SEQ ID NO: 53) (FIG. 57C) V-light chains. The putative reverted unmutated ancestor sequence was used as template for both the V-heavy and the CH01-CH04 V-light alignments. Since CH05 has an unrelated V.kappa.1.about.6 chain, it is shown separately.

[0069] FIG. 58. Phylogenetic tree of the V-heavy chains of the CH01-CH05 monoclonal antibodies.

[0070] FIG. 59. Alignment of the inferred putative reverted unmutated ancestor antibodies. The alignment of all the putative reverted unmutated ancestor antibodies inferred by applying the V-heavy chains are separated from the V-light chains by ".about. .about. .about.." FIG. 59 discloses SEQ ID NOS 54-78, respectively, in order of appearance.

[0071] FIG. 60. Binding of CH01, CH02, CH03 quarternary broad neutralizing antibodies to A244 gp120.

[0072] FIG. 61. Binding of reverted unmutated ancestors of CH01, CH02, CH03 quarternary broad neutralizing antibodies to A244 gp120.

[0073] FIG. 62. PG9 and PG16 bind to both A244 gp120 and 6420 T/F gp140.

[0074] FIG. 63. CH01 monoclonal antibodies decreased by binding affinity to A244gD-gp120 envelope compared to A244gD+gp120 envelopes.

[0075] FIG. 64. Forty-eight percent anti-gD IgA vaccine response (99 subjects).

[0076] FIG. 65. Eight-one percent anti-gD IgG vaccine response (99 subjects).

[0077] FIG. 66. Potential relevance of gD immunogenicity. FIG. 66 discloses SEQ ID NOS 79-80, respectively, in order of appearance.

[0078] FIG. 67. HEP-2 binding

[0079] FIG. 68. Effect of kifunensine treatment on the ability of CH01 to mediate neutralization

[0080] FIG. 69. Superimposition of the sequence of CH01 (here called 1-27-G2) with the PG16 Fab.

DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention relates to a method of inducing the production in a subject (e.g., a human subject) of broadly neutralizing antibodies against HIV-1. The method comprises administering to the subject a non-HIV-1 antigen that binds to a germline B cell receptor, the non-HIV-1 antigen being administered in an amount and under conditions such that intermediate clones of B cells are produced that secrete antibodies that cross-react with HIV-1 Env. The method further comprises administering to the subject an HIV-1 antigen in an amount and under conditions such that naive B cells or their B cell intermediate clones are produced that secrete the broadly neutralizing anti-HIV-1 antibodies. It is likely that, for some epitopes on gp120, there will be rare naive B cells capable of binding to those epitopes while, for other epitopes, naive B cells that can give rise to broadly neutralizing antibodies will not bind Env and will need to be stimulated by additional non-Env epitopes. Roadblocks to inducing broadly neutralizing antibodies are described in FIG. 15 and the present strategy for overcoming those roadblocks is described in FIG. 16A.

[0082] Non-HIV-1 antigens suitable for use in the invention include host and/or foreign antigens. Non-HIV-1 antigens include, for example, lipids, such as cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphotidylinositol, sphingomyelin, and derivatives thereof, e.g., 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine (DOPE), or fragments thereof. Use of hexagonal II phases of phospholipids can be advantageous and phospholipids that readily form hexagonally packed cylinders of the hexagonal II tubular phase (e.g., under physiological conditions) are preferred, as are phospholipids that can be stabilized in the hexagonal II phase. (See Rauch et al, Proc. Natl. Acad. Sci. USA 87:4112-4114 (1990); Aguilar et al et al, J. Biol. Chem. 274: 25193-25196 (1999)). Other suitable non-HIV-1 antigens include, for example, phycoerythrin (PE), C-phycocyanin (C-PC), or other phycobiliprotein, apoferritin, and anerobic or aerobic gut flora or component(s) thereof (for example, the 520Kd antigen (or the RNA polymerase holoenzyme or the RNA polymerase core protein, or subunit thereof, such as the a subunit of RNA polymerase core protein or portion thereof comprising the epitope to which mAb HV00276 binds), or the 60Kd or 50Kd antigen). The data presented in Example 2 indicates that mAb HV00276 binds to the .alpha. subunit of E. coli RNA polymerase core protein. The sequence homology is high between the .alpha. subunit of E. coli RNA polymerase core protein and a homologs from other bacteria (e.g., B. subtilis, S. dysenteriaea, S. enterica, M. tuberculosis, H. pylori and H. influenza) and eukaryotes (e.g., human and mouse proteins related to S. cerevisiae Rpb3 and Rpb11) (Zhang and Darst, Science 281:262-266 (1998)). Accordingly, the invention includes the use of the 520Kd antigen (or subunit thereof, such as the .alpha. subunit of RNA polymerase core protein or portion thereof comprising the epitope to which mAb HV00276 binds) from eukaryotes and from bacteria in addition to E. coli. (See, for example, E. coli RNA polymerase .alpha. subunit: NP_289856 (gi/15803822); S. dysenteriaea: YP_404940 (gi:82778591); H. influenzae: NP_438962 (gi:16272744); Rpb3: Swiss-Prot: P37382.2; Rpb3 (Homo sapiens): NP_116558.1 (gi:14702171).)

[0083] Kynureninase (KYNU) is a member of the family of pyridoxal 5'-phosphate (PLP)-dependent enzymes known as the aspartate aminotransferase superfamily. Eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-1-kynurenine to produce 3-hydroxyanthranilate and 1-alanine. The cloning, expression, purification, characterization and crystallization of Homo sapiens KYNU has been reported (Lima et al, Biochemistry 46(10):2735-2744 (2007). As described in Example 3 below, KYNU carries the core 2F5 epitope in its conserved H3 domain.

[0084] Based on the data provided in Example 3, it is anticipated that this endogenous ligand is responsible for tolerizing B and T lymphocytes and thereby inhibiting the production of effective immune responses against HIV-1 in humans administered HIV-1 gp41 MPER epitope peptides. The invention provides, in one embodiment, methods of effecting immunization against HIV-1 comprising administering cross-reactive antigens that break this tolerance specifically, that is, without affecting tolerization against other, irrelevant self antigens. Suitable antigens include, for example, the recombinant KYNU molecule expressed in CHO or 293T cells with the ELDKWA (SEQ ID NO: 2) sequence or a mutant gp41 or KYNU sequence with the ELEKWA (SEQ ID NO: 3) sequence (ELEKWA (SEQ ID NO: 3) is not present in human proteins and thus is not expected to be tolerizing). Other immunogens that can be used include transmitted/founder or wildtype chronic envelope gp140s or gp160s or MPER peptides in liposomes with either the ELEKWA (SEQ ID NO: 3) or the ELDKWA (SEQ ID NO: 2) sequence. Immunogens with the ELDKWA (SEQ ID NO: 2) sequence are, advantageously, administered with strong adjuvants, such as squalene based monophosphosphoryl lipid A, oligonucletides (oCpGs) and R848 (TRL-7/8 agonist). Liposomes with these TLR agonists and IFN.alpha. can also be used. (See also comments below.)

[0085] HIV-1 antigens suitable for use in the invention include membrane-proximal external region (MPER) antigens (Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004), Stiegler and Katinger, J. Antimicrob. Chemother. 512:757-759 (2003), Zwick et al, Journal of Virology 79:1252-1261 (2005), Purtscher et al, AIDS 10:587 (1996)) and variants thereof, for example, variants that confer higher neutralization sensitivity to MPER Mabs 2F5 and 4E10 or to other broadly neutralizing Envs, such as the MPER mutant Env peptide lipid complex containing a L669S mutation in the MPER (Shen et al, J. Virology 83:3617-25 (2009)). Preferred immunogens include those shown in FIGS. 25 and 26, as well as FIGS. 16B, 16C, FIG. 17, FIG. 18 and FIG. 20. In another preferred embodiment, the variant is a MPER epitope peptide with an L669S mutation that confers higher neutralization sensitivity to MPER mAbs 2F5 and 4E10 (Shen et al, J. Virology 83: 3617-25 (2009)).

[0086] HIV-1 antigens suitable for use in the invention also include transmitted founder HIV-1 Envs, or fragments thereof. These fragments can be representative of portions of the CD4 binding site of gp120 (Chen et al, Science 362(5956):1123-7 (2009)), MPER sequences, portions of gp120 incorporating the V2, V3 regions of gp120 (Walker et al, Science 326(5950):285-9 (2009) Epub 2009 Sep. 3), etc (e.g., see the sequences for 1086, 089, 6240, 040_C9 and 63521 set forth in FIGS. 27 and 28). Preferred Env antigens include the Malawi 1086 clade C, 6321 and the US clade B 040_C9 gp140 oligomers (FIGS. 17 and 18) (Keele et al, Proc. Natl. Acad. Sci. USA 105:7552-7 (2008)) produced as previously described (Liao et al, Virology 30:268-282 (2006)), which have induced in guinea pigs considerable breadth in neutralizing antibodies (FIG. 19A), mixed with the clade B JRFL gp140 Env, or fragment thereof, that selectively expresses the MPER neutralizing epitopes (see FIG. 28). The JRFL gp140 Env oligomer (FIGS. 19B, 20, 21A and 21B) constitutively binds the 2F5 mAb. The JRFL oligomer deglycosylated using 500U of PNgase endoglycosidase (New England BioLabs, Ipswich, Mass.) has enhanced binding of 2F5 and new binding of the 4E10 mAb (exposure of the 4E10 epitope on gp41) (FIGS. 21A and 21B). The enhanced binding of 4E10 to deglycosylated JRFL is also shown in surface plasmon reasonance (SPR) analysis in FIG. 22.

[0087] The method of the invention can be effected by administering to the subject a prime immunization comprising a non-HIV-1 immunogen followed by one or more boosts of an HIV-1 Env antigen. As pointed out above, suitable non-HIV-1 immunogens include lipids (e.g., cardiolipin, phosphotidylserine, or other anionic lipid), components of anaerobic or aerobic gut flora bacteria, phycobiliproteins (e.g., PE) and KYNU or fragment thereof. As also pointed out above, suitable HIV-1 Env antigens include transmitted founder Env 1086.C from Malawi, 089.C from Malawi, 040_C9 from the U.S. and 63521 from a Clade B acute HIV-1 infected U.S. patient. Both the primes and the boosts suitable for use in the present method can comprise both non-HIV-1 and HIV-1 immunogens. Prime/boost regimes can be readily optimized by one skilled in the art. DNA sequences encoding proteinaceous components of such regimens can be administered under conditions such that the proteinaceous component is produced in vivo.

[0088] As described in Example 5 below, 5 clonally related B cells have been isolated from a single patient that produce broadly neutralizing antibodies (CH01 through CH05). Possible reverted unmutated ancestors of the clonally-related antibodies have been inferred and expressed as real antibodies. The phylogenetic tree of these antibodies has been reconstructed. Both the natural and inferred ancestor antibodies have been characterized for their ability to bind a panel of HIV envelope proteins and to neutralize a panel of HIV isolates. It is important to note that the reverted unmutated ancestors (RUAs) bind to A244gD+ envelope. Therefore, such envelope, or other envelopes described to be neutralized by the RUAs, can be used as the "prime" in a preferred vaccine strategy of the invention. In accordance with this strategy, the "boost" can be effected, for example, using envelopes that are bound by the mature antibodies described herein. A further "boost" can be effected, for example, with 6420 or 63521 (or other protein, peptide or polypeptide that binds).

[0089] When a DNA prime or boost is used, suitable formulations include a DNA prime and a recombinant adenovirus boost and a DNA prime and a recombinant mycobacteria boost, where the DNA or the vectors encode, for example, either HIV-1 envelope or a proteinaceous non-HIV1-1 antigen, such as a gut flora or KYNU component. Other combinations of these vectors can be used as primes or boosts, either with or without HIV-1 antigen and/or non-HIV-1 antigen.

[0090] In accordance with the invention, the non-HIV-1 antigen can be present in a liposome with the HIV-1 Env antigen and one or more adjuvants. Alternatively, the non-HIV-1 antigen can be conjugated, for example, using a hetero-bifunctional agent such as DSSP, to the HIV-1 Env antigen and formulated with one or more adjuvants.

[0091] Liposomes expressing MPER antigens (Dennison, et al, J. Virology 83:10211-23 (2009)) with or without Toll Like Receptor (TLR) agonists have been described (see, for example, WO 2008/127651). Gp41 intermediate state protein (FIG. 23) has been described by (Frey et al, Proc. Natl. Acad. Sci. USA 105-3739-44 (2008)). The gp41 intermediates can be formulated with liposomes (FIGS. 24A and 24B) to form a stable immunogens that bind well to 2F5 and 4E10 (FIG. 25). Gp41 MPER immunogens of the invention can be adjuvanted by incorporating, for example, monophosphorylipid A (MPL-A) (Avanti Polar Lipids, Alabaster, Ala.) and a TLR 9 agonist, such as oCpGs 10103 (5'-TCGTCGTTTTTCGGTCGTTTT-3') (SEQ ID NO: 4) and R848 TLR 7 agonist (Enzo Life Sciences, Farmingdale, N.Y.) (FIG. 26). In addition, cytokine stimulators of B cell class switching, such as BAFF (BLYS) and/or APRIL (He et al, Immunity 26:812-26 (2007); Cerutti and Rescigno, Immunity 28: 740-50 (2008)) can be incorporated into the liposomes for optimal B cell stimulation.

[0092] Liposomes suitable for use in the invention include, but are not limited to, those comprising POPC, POPE, DMPA (or sphingomyelin (SM)), lysophosphorylcholine, phosphatidylserine, and cholesterol (Ch). While optimum ratios can be determined by one skilled in the art, examples include POPC:POPE (or POPS):SM:Ch or POPC:POPE (or POPS):DMPA:Ch at ratios of 45:25:20:10. Alternative formulations of liposomes that can be used include DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) (or lysophosphorylcholine), cholesterol (Ch) and DMPG (1,2-dimyristoyl-sn-glycero-3-phoshpho-rac-(1-glycerol) formulated at a molar ratio of 9:7.5:1 (Wassef et al, ImmunoMethods 4:217-222 (1994); Alving et al, G. Gregoriadis (ed.), Liposome technology 2.sup.nd ed., vol. III CRC Press, Inc., Boca Raton, Fla. (1993); Richards et al, Infect. Immun. 66(6):285902865 (1998)). The above-described lipid compositions can be complexed with lipid A and used as an immunogen to induce antibody responses against phospholipids (Schuster et al, J. Immunol. 122:900-905 (1979)). A preferred formulation comprises POPC:POPS:Ch at ratios of 60:30:10 complexed with lipid A according to Schuster et al, J. Immunol. 122:900-905 (1979). The optimum ratio of peptide to total lipid can vary, for example, with the peptide and the liposome.

[0093] A variety of adjuvants can be used in the present invention (including those noted above). The peptide-liposome immunogens and the conjugates described above can be formulated with, and/or administered with, adjuvants such as squalene-based adjuvants (Kaldova, Biochem. Biophys. Res. Communication, Dec. 16, 2009 E-pub ahead of print) and/or TLR agonists (e.g., a TRL 3, TRL 5, TRL4, TRL9 or TRL7/8 agonst, or combination thereof) that facilitate robust antibody responses (Rao et al, Immunobiol. Cell Biol. 82(5):523 (2004)). Other adjuvants that can be used include alum and Q521. Oligo CpGs in an oil emulsion such as Emulsigen (an oil in water emulsion) (Tran et al, Clin. Immunol. 109(3):278-287 (2003)) can also be used. Additional suitable adjuvants include those described in Ser. No. 11/302,505, filed Dec. 14, 2005, including the TRL agonists disclosed therein. (See also Tran et al, Clin. Immunol. 109:278-287 (2003), US Appln Nos. 20030181406, 20040006242, 20040006032, 20040092472, 20040067905, 20040053880, 20040152649, 20040171086, 20040198680, 200500059619). Immune response enhancing TLR ligands, such as Lipid A, oligo CpG and R-848 can be formulated individually or in combination into liposomes that have HIV-1 Env conjugated in them.

[0094] Liposomes loaded with strong adjuvants (e.g., potent TLR agonists) are examples of immunogens that can be used to overcome peripheral deletion and/or anergy of B cells that do get driven to make polyreactive neutralizing antibodies.

[0095] Transmembrane domain anchoring of HIV-1 gp41 peptides to liposomes can be used to achieve functional epitope display. The transmembrane domain of HIV-1 gp41 can be used to anchor the peptide into liposomes comprising synthetic lipids. Induction of trimerization of the TMD can facilitate formation of trimeric forms of gp41 MPER. Alternatively, His-tagged (c-terminus end) versions of the Env gp140 can be anchored into liposomes as described for an intermediate form of HIV-1 gp41 (gp41-inter).

[0096] The mode of administration of the non-HIV-1 immunogen and/or HIV-1 protein/polypeptide/peptide, or encoding sequence, can vary with the immunogen, the patient and the effect sought, similarly, the dose administered. Typically, the administration route will be intramuscular, intravenous, intraperitoneal or subcutaneous injection. Additionally, the formulations can be administered via the intranasal route, or intrarectally or vaginally as a suppository-like vehicle. Generally, the liposomes are suspended in an aqueous liquid such as normal saline or phosphate buffered saline pH 7.0. Optimum dosing regimens can be readily determined by one skilled in the art. The immunogens are preferred for use prophylactically, however, their administration to infected individuals may reduce viral load.

[0097] The human monoclonal antibodies (hu mAb) 2F5 and 4E10 bind with high specificity and nanomolar (nM) affinities to polypeptides that correspond to the HIV-1 gp41 MPER. Both hu mAb also react with discrete human and mouse cellular antigens as determined by immunofluorescence microscopy and western blotting. These properties indicate that 2F5 and 4E10 are ideal for the isolation of cellular proteins, including denatured forms and polypeptides, biochemically extracted from mammalian cells and recovered by standard immunoprecipitation methods. The same properties of 2F5 and 4E10 make them suitable for the identification of extracted cellular proteins/polypeptides by the standard methods of mass spectroscopy. Briefly, immunoprecipitated cellular proteins/polypeptides specifically bound to 2F5 or 4E10 can be subjected to enzymatic digestion and the mass and charge of the resulting fragments used to identify the parental molecule(s).

[0098] Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow (see also Maksyutov et al, J. Clin. Virol. December; 31 Suppl 1:S26-38 (2004), Haynes et al, Science 308:1906 (2005), Gurgo et al, Virology 164:531-536 (1988), U.S. Pat. No. 7,611,704, U.S. application Ser. No. 11/812,992, filed Jun. 22, 2007, U.S. application Ser. No. 11/785,077, filed Apr. 13, 2007, PCT/US2006/013684, filed Apr. 12, 2006, PCT/US04/30397 (WO2005/028625), WO 2006/110831, WO 2008/127651, U.S. Published Application Nos. 2008/0031890 and 2008/0057075, U.S. application Ser. No. 11/918,219, filed Dec. 22, 2008, U.S. Prov. Appln. No. 60/960,413, filed Feb. 28, 2007, and U.S. Prov. Appln. Nos. 61/166,625, 61/166,648 and 61/202,778, all filed Apr. 3, 2009, U.S. Prov. Appln. No. 61/282,526, filed Feb. 25, 2010, U.S. Prov. Appln. No. 61/344,457, filed Jul. 27, 2010, U.S. Prov. Appln. Client File No. 01579-1597, filed Aug. 25, 2010, PCT/US2010/01018, PCT/US2010/030011, and PCT/US2010/01017 the entire contents of which are incorporated herein by reference).

EXAMPLE 1

Experimental Details

[0099] Acute HIV-1 Infected Patients. The patients selected for study were from 17 to 30 days following transmission with the dates of transmission estimated from patient history and Fiebig classification (Fiebig et al, AIDS 17:1871-1879 (2003)). Patients 065-0 and FIKE were Fiebig Stage 1, while patients 068-9, 684-6 and MCER were Fiebig stage 2.

[0100] Control Subjects. Single plasmablast/plasma cell sorts were performed on bone marrow, leukapheresis or peripheral blood mononuclear cells (PBMC) of uninfected subjects as well as those vaccinated with trivalent inactivated (TVI) influenza vaccine (FLUZONE.RTM. 2007 or 2008). Those immunized with TVI were studied 7 days after immunization (Liao et al, J. Virologic Methods 158:171-9, (2009); Wrammert et al, Nature 453:667-71 (2008); Smith et al, Nature Protocols 4:372-84 (2009)).

[0101] Flow Sorting Strategy. PBMC, leukapheresis or bone marrow samples were reacted with anti-B cell antibodies as previously described (Liao et al, J. Virologic Methods 158:171-9 (2009)). Wrammert et al (Nature 453:667-71 (2008)) have shown that the cells that are antibody secreting cells in human PBMC are those that are within the CD19.sup.+, CD38.sup.hi+, IgD.sup.-, CD20.sup.lo+/- B cells. Thus, in both acute HIV infection (AHI) and in influenza vaccine vaccinated controls, to isolate single antibody secreting plasmablasts/plasma cells, CD19.sup.+, CD38.sup.hi+, IgD.sup.-, CD20.sup.lo+/- cells were sorted by flow cytometry into single 96 well plates containing RNA extraction buffer as described (Liao et al, J. Virologic Methods 158:171-9 (2008); Wrammert et al, Nature 453:667-71 (2008)). As positive controls for definition of successful isolation of the correct plasmablast/plasma cell population, the same population was isolated from day 7 after trivalent influenza vaccine (FLUZONE.RTM. 2007 or 2008) vaccines. It was demonstrated that, as expected, 75% of those sorted cells were indeed influenza specific antibodies (Wrammert et al, Nature 453:667-71 (2008)).

[0102] Identification and expression of the transmitted/founder envelope. The transmitted/founder Env of patients 684-6 and FIKE were identified by single genome amplification and Env gene sequencing as previously described (Keele et al, Proc. Natl. Acad. Sci. USA 105:7552-7 (2008)). Env gp140C (gp120/41 cleavage site mutated), gp120 and gp41 proteins were expressed by transient transfections of 293T cells as described (Liao et al, J. Virologic Methods 158:171-9 (2008)).

[0103] PCR amplification of plasmablast/plasma cell immunoglobulin VH and VL genes. The VH and VL Ig chains of sorted B plasmablast/plasma cells were isolated by single cell PCR and recombinant antibodies produced as described (Liao et al, J. Virologic Methods 158:171-9 (2009); Wrammert et al, Nature 453:667-71 (2008); Smith et al, Nature Protocols 4:372-84 (2009)).

[0104] Sequencing, sequence annotation, quality control, and data management of Ig VH and VL sequences. All PCR products of Ig VH and VL genes were purified using a Qiagen (Valencia, Calif.) PCR purification kit and sequenced in forward and reverse directions using an ABI 3700 instrument and BigDye.RTM. sequencing kit (Applied Biosystems, Foster City, Calif.). Base calling for each sample is done using Phred (Ewing et al, Genome Res. 8:175-85 (1998); Ewing and Green, Genome Res. 8:186-94 (1998)). The forward and reverse strands of the antibody genes are assembled to one final nucleotide sequence using a novel assembly algorithm based on the quality scores at each position (Kepler et al, submitted). The estimated PCR artifact rate was 0.28 or approximately 1 PCR artifact per 5 genes amplified. The isotype of the immunoglobulin is determined by a local alignment algorithm (Smith and Waterman, J. Mol. Biol. 147:195-7 (1981)). The germline rearrangement of the quality assured antibody sequence is determined using SoDA (Volpe et. al, Bioinformatics 22:438-44 (2006)). Genomic information derived from SoDA, such as gene segment usage, somatic mutations and CDR3 regions, are stored in an ORACLE database for easy access.

[0105] To determine if antibodies from the same subject are clonally related, the following 3 criteria were utilized. First, the heavy chain of the antibodies in question must use the same VH and JH gene segments. Due to the length and high mutation in the D segment, these are more difficult to identify. Thus, similarity of D segments is not used as criteria for clonal relatedness. Similarly, both light chains must use the same V.kappa./V.lamda. and J.kappa./J.lamda.. Second, the heavy chains of the antibodies in question must have the same CDR3 length. This also applies to light chains. Third, the nucleotide sequence of the CDR3 of the heavy chains must be 70% identical. The same applies to the CDR3 of the light chain. Antibodies that adhere to these three criteria are labeled as being clonally related. Maximum Likelihood trees were constructed to determine the phylogenetic relationship between the clones using the PHYLIP 3.63 package (Felsenstein, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360:1427-34 (2005)) using the inferred germline from SoDA as the root. The ancestral sequences were also inferred using the same package.

[0106] Design and generation of inferred germline and intermediate antibodies. For each member of an antibody clonal family, Maximum Likelihood analysis was used to infer the germline antibody precursor as well as multiple antibody intermediate forms (Felsenstein, J. Mol. Evol. 17: 368-76 (1981); Volpe et al, Bioinformatics 22:438-44 (2006)). These VH and VL genes were synthesized (GeneScript, Piscataway, N.J.) and expressed as IgG1 mAbs by recombinant techniques as above.

[0107] Expression of V.sub.H and V.sub.L as recombinant mAbs. The isolated Ig V.sub.H and V.sub.L gene pairs were assembled by PCR into the linear full-length Ig heavy- and light-chain gene expression cassettes for production of recombinant mAbs by transfection in human embryonic kidney cell line, 293T (ATCC, Manassas, Va.) using the methods as described (Liao et al, J. Virol. Methods 158:171-9 (2009)). The purified PCR products of the paired Ig heavy- and light-chain gene expression cassettes were co-transfected into 80-90% confluent 293T cells grown in 6-well (2 .mu.g of each per well) tissue culture plates (Becton Dickson, Franklin Lakes, N.J.) using PolyFect (Qiagen, Valencia, Calif.) and the protocol recommended by the manufacturer. Six to eight hours after transfection, the 293T cells were fed with fresh culture medium supplemented with 2% fetal calf serum (FCS) and were incubated at 37.degree. C. in a 5% CO.sub.2 incubator. Culture supernatants were harvested three days after transfection and quantified for IgG levels expressed and screened for antibody specificity. For future characterization of select antibodies identified through screening assays, the linear Ig heavy and light chain gene constructs were cloned into pcDNA 3.3 for production of purified recombinant mAbs using standard molecular protocols.

[0108] For production of purified recombinant mAbs derived from the isolated VH and VL genes and the inferred germline and intermediate precursor antibody sequences, 293T cells cultured in T175 flasks were co-transfected with the heavy and light chain Ig gene-containing plasmids using PolyFect (Qiagen, Valencia, Calif.), cultured in DMEM supplemented with 2% FCS. Recombinant mAbs were purified from culture supernatants of the transfected-293T cells using anti-human Ig heavy chain specific antibody-agarose beads (Sigma, St. Louis, Mo.).

[0109] Screening for antibody specificity by ELISA and Luminex assays. Concentration of recombinant mAbs in the supernatants were determined using the method as described (Liao et al, J. Virol. Meth. 158:171-179 (2009)). Specificity of the expressed recombinant mAb were assayed for antibody reactivity to HIV-1 antigens and to a panel of non-HIV-1 antigens. HIV antigens included Env peptides gp41 immunodominant region (RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNK) (SEQ ID NO: 5), gp41 MPER region (QQEKNEQELLELDKWASLWN) (SEQ ID NO: 6), HIV-1 MN gp41 (Immunodiagnostics, Woburn, Mass.), HIV-1 group M consensus gp120 (Liao et al, Virology 353:268-82 (2006)), HIV-1 group M consensus gp140 CFI (Liao et al, Virology 353:268-82 (2006)), p66 (Worthington Biochemical, Lakewood, N.J.), p55 (Protein Sciences, Meriden, Conn.), p31 (Genway, San Diego, Calif.), nef (Genway, San Diego, Calif.), tat (Advanced BioScience, Kensington, Md.) and AT-2 inactivated HIV-1 ADA virions (Rutebemberwa et al, AIDS Res. Human Retrovirol. 23:532-42 (2007)); gift of Jeffrey Lifson, NIH, NCI, Frederick Cancer Research Facility). In addition, 684-6 mAbs were assayed against autologous gp140, gp120 and gp41, and FIKE mAbs were assayed against autologous gp140 and gp120. Non-HIV-1 antigens included trivalent influenza vaccine 2007 (FLUZONE.RTM. 2007), recombinant influenza HA protein from H1 A/Solomon Islands/03/2006 (Protein Sciences Corp. Meriden, Conn.), tetanus toxiod (Calbiochem, San Diego, Calif.), HEP-2 cells (Inverness Medical Professional Diagnostics, Princeton, N.J.), cardiolipin (Avanti Polar Lipids, location (Alabaster, Ala.) (Haynes et al, Science 308:1906-8 (2005)) and lipid A (Avanti Polar Lipids, Alabaster, Ala.). Whole cell lysates of anaerobic and aerobic bacterial extracts termed as gut flora were prepared as described below. Briefly, bacteria were inoculated from 4 stool specimens from patients and grown on agar plates under anaerobic or aerobic conditions at 30.degree. C. Confluent bacteria were harvested, washed twice with phosphate-buffered saline (PBS) and treated with a commercially available bacterial protein extraction reagent (Pierce, Rockford, Ill.). The resulting extracts were filtered with a 0.22 .mu.m filter and stored at -80.degree. C. until use (Kawatsu et al, J. Clin. Microbiol. 46:1226-31 (2008)). Assays against FLUZONE.RTM., influenza HA, gp41 immunodominant and MPER regions, as well as gut flora whole cell lysates, were performed by both ELISA (Tomaras et al, J. Virology 82:12449-63 (2008)) and Luminex bead assays (Tomaras et al, J. Virology 82:12449-63 (2008)). Assays against tetanus toxoid, cardiolipin (Sigma, St Louis, Mo.), killed Cryptococcus and Candida albicans were ELISA Assays for reactivity with Hep-2 epithelial cells were indirect immunofluoresence assays (Mietzner et al, Proc. Natl. Acad. Sci. USA 105:9727-32 (2009)).

[0110] Surface Plasmon reasonance (SPR) analysis of antibody reactivity. SPR binding assays were performed on a BIAcore 3000 (BIAcore Inc, Piscattaway, N.J.) maintained at 20.degree. C. HIV-1 gp41 or oligomeric gp140 proteins (Con S gp140, autologous Env gp140) were immobilized on a CM5 sensor chip by standard amine coupling as previously described (Alam et al, J. Immunol. 178:4424-35 (2007)). Human mAbs were captured on anti-human Fc antibody coupled surfaces and then each human mAbs were captured to about 200-500 RU. Specific binding responses of mAb binding were obtained following subtraction of non-specific binding on control surfaces (HIV-1 gp120 for Env immobilized surfaces and human IgG, IS6, for mAb captured surfaces). Rate constants were measured using the bivalent analyte model (to account for the avidity of bivalent Ig molecules) and global curve fitting to binding curves obtained from mAb titrations. MAbs were injected at 30 .mu.L/min for 2-6 min and glycine-HCl pH 2.0 and surfactant P20 (0.01%) were used as the regeneration buffer.

Results

[0111] Influenza vaccination. Clones of antibodies from influenza vaccinated subjects derived from single cell sorted plasma cells/plasmablasts were studied and the response was found to be highly clonal. The clones members almost all reacted with the influenza antigen tested. FIG. 1 shows a representative influenza antibody clone against H1 Soloman Islands hemagglutinin. A total of 450 antibodies were isolated from plasma cells/plasmblasts of three influenza vaccinated subjects and, of these, 57.7% were influenza-specific. Of all the 265 antibodies isolated from influenza infected subjects, twenty independent clones of clonally related antibodies were identified, among which, 115 antibodies (92%) reacted with influenza antigens.

[0112] Clonal antibody response in acute HIV infection. In contrast to influenza vaccination, where .about.75% of plasma cells/plasmablasts were influenza specific, out of a total of 1074 recombinant antibodies that have been isolated from plasmablasts/plasma cells of 5 AHI patients, 89 or 8.3% expressed antibodies (range 3.3% to 13.4%) were HIV-1 specific, while the majority of the remainder of the mAbs either were against non-HIV antigens (.about.6%) or had unknown specificity (882 or 82.1%). With the panel of non-HIV-1 related antigen assays, it was possible to demonstrate high affinity antibodies to Hep-2 epithelial cells (27 or 2.5%), gut flora (5 or 0.5%), cardiolipin (4 or 0.4%), influenza (9 or 0.8%), Cryptococcus (4 or 0.4%), Candida albicans (2 or 0.2%), and tetanus toxoid (8 or 0.7%). An additional 38 or 3.5% reacted with at least 2 of these antigens. Three of the patients had lipid A and one patient had gut flora antibodies suggesting the very early onset of gut damage, microbial translocation and induction of anti-lipid A and gut flora antibodies. Remarkably, none of these early AHI patients had any mAbs detected with HIV-1 specificities other than gp41 within days 17-30 after HIV transmission.

[0113] It was previously reported that consensus Envs were equal to autologous Envs in detecting the AHI response to gp41 (Tomaras et al, J. Virology 82: 12449-63 (2008)). However, to rule out the possibility that responses were being missed in AHI B cell analysis, the mAbs from 684-6 and FIKE were screened with their autologous recombinant gp140 Envs. In general, the response to the autologous gp140 envs was much less than to the clade B gp41.

[0114] Thus, the initial plasmablast/plasma cell repertoire response to the transmitted/founder virus, like the plasma antibody response (Tomaras et al, J. Virol. 82:12440-63 (2008)), was focused on Env gp41 epitopes. In addition, HIV-1 activates and drives to terminal differentiation preexisting memory B cells from previous vaccination or infectious agent antigens, such as Cryptococcus, Candida albicans, and tetanus toxoid. Moreover, in the course of AHI, polyreactive clones of Hep-2 cell autoreactive B cells are triggered to join the initial plasmablast/plasma cell response.

[0115] Analysis of antibody clones within the AHI plasmablast/plasma cell repertoire. In general, there few clones isolated from the AHI plasmablast/plasma cell repertoire compared to the reported plasmablast/plasma cell repertoires induced by influenza vaccination (Wrammert et al, Nature 453:667-72 (2009)) or the memory B cell repertoire of gp140+ B cells in subjects with broad neutralizing antibody activity in plasma (Scheid et al, Nature 458:636-40 (2009)). In chronic HIV-1 infection in six patients with broad neutralizing antibodies, Scheid et al (Nature 458:636-40 (2009)) found the number of B-cell clones varied among patients from 22 to 50 in 502 antibodies isolated from those six patients.

[0116] In the study of AHI, only 8 clones of antibodies were found in 1074 mAbs isolated from 5 AHI patients. These included three clones of antibodies that reacted with gp41 among 6 independent clones of antibodies identified in one of AHI patients. Of interest, of all 52 clonal members of the 3 AHI gp41 clones, only 17 (37%) reacted with gp41. This is in contrast to 94% of influenza-reactive influenza clone members.

[0117] FIG. 2 shows AHI clone 684-6B--a remarkable VH3-7, DH1-26, JH5, VK1-39, JK4, IgG3 mutated clone with 52 members, with no unmutated members. Out of the 57 antibodies, only 4 (8%) reacted with gp41.

[0118] Analysis of the gp41 reactivity with clone inferred germline and intermediate antibodies. It was reasoned that either HIV-1 gp41 was reacting with the germline B cell receptor of naive B cells and was stimulating low affinity clones with poor antigen drive, or that gp41 may cross-react with pre-existing clones of memory B cells and enjoin clonal members to undergo simultaneous gp41 and self antigen drive. To distinguish between these two possibilities, Maximum Likehood analysis was used to infer the germline unmutated antibody and partially mutated clone intermediates were used to determine their reactivity with gp41 (FIG. 3). To determine where in the clone development reactivity with gp41 was acquired (i.e., germline VH+VL or later intermediates), inferred germline and clone member intermediates were assayed for reactivity with clade B gp41, autologous gp140 and group M consensus gp140 (FIG. 4). It was found that reactivity of clone 684-6B was acquired at the second intermediate precursor antibody (FIGS. 4 and 5).

[0119] The next question asked was whether the reactivity with gp41 represented antigen drive by gp41. FIG. 6 shows more inferred intermediate antibody clones were produced in mg quantities and analyzed for the dissociation constants (Kd) of antibody binding to gp41. FIG. 7 shows a heat map plot with the dissociation constants plotted as log 10 of the Kds, and demonstrates that, indeed, as the intermediates progress to actual isolated antibodies, there is progression of affinity maturation for binding to gp41.

[0120] Given the induction of polyreactive non-HIV-1 gp41 clones during AHI, the next question asked was whether clone 684-6B members were polyreactive by reactivity with cardiolipin and Hep-2 epithelial cells. In the Hep-2 indirect immunofluoresence assay, reactivity of clone 684-6B was acquired at the same inferred intermediate precursor stage as gp41 reactivity (FIG. 8). All clone members of 684-6B reacted with cardiolipin, including the germline unmutated antibody, and while Hep-2 reactivity waxed and waned during clone development, reactivity with cardiolipin was relatively stable throughout the intermediates until the end clones 307 and 350. The polyreactivity of the germline and other clone members with cardiolipin strongly suggests that the initial antibody response to HIV is derived from HIV gp41 stimulating a preexisting, polyreactive clone of natural antibodies and gp41 recruits clones of B cells to become polyreactive gp41 clones as soon as the original clone acquires cross reactivity to gp41 by somatic hypermutations. This finding has considerable ramifications to HIV vaccine design.

[0121] The nature of the germline reactivity to non-HIV-1 antigens. Given the surprising result of the acquisition of reactivity of the 684-6B clone not in the germline antibody of each clone but in inferred clone intermediates, an effort was made to identify host antigens against which the germline might react to identify likely origins of the antibody clones activated in HIV.

[0122] It was hypothesized that because there is early gut microbial translocation in the gut due to AHI and because much of the initial antigenic stimulation in AHI comes at mucosal surfaces, the initial antibody response may in some manner be tied to or related to the gut microbial antibody response. To study this, a determination was made as to whether there were measurable reactivity of the clonal antibodies and inferred germline and inferred intermediates from 684-6B clone to the whole cell lysates of anerobic and aerobic gut flora. In addition, EBV transformation was used to isolate a panel of pentameric IgM mAbs from intestine, bone marrow or blood of AHI or uninfected subjects.

[0123] First, a series of IgM antibodies was isolated from AHI and two from uninfected subjects that were either gp41 reactive or gp41 non-reactive. The question asked was whether the IgMs that were reactive with gp41 also were reactive with gut flora. Table 1 shows that, indeed, all the mAbs that were gp41 reactive were also reactive with gut flora antigens while those mAbs that were not reactive with gp41 were not gut flora reactive.

TABLE-US-00001 TABLE 1 All HIV-1 Env gp41 IgM Mabs Isolated from Infected or Uninfected also Bind to Either Anerobic or Aerobic Gut Bacterial Whole Cell Lysates HIV-1 Anerobic Gut Aerobic Gut MAb Env gp41 Bacteria WCL Bacteria WCL Source of Mab Reactivity in Luminex Units 21B10 173 272 1012 AHI intestine 2C3 148 210 591 AHI intestine F3 177 671 2237 AHI intestine F8 1023 372 5433 AHI intestine 1E7 17153 259 133 AHI bone marrow 2B9 24886 742 347 AHI bone marrow ALL8 13031 1816 1584 AHI intestine C14-2 2500 172 >80 uninfected intestine C08 3673 241 >80 uninfected blood XM-1 <80 <80 <80 uninfected blood XM-2 <80 <80 <80 AHI intestine XM-3 <80 <80 <80 AHI intestine AHI = acute/early HIV-1 infection. Mab = monoclonal antibody. WCL = whole cell lysate. <80 = no reactivity over background in Luminex assay with gp41 or gut flora whole cell lysates.

[0124] Remarkably, when the germline and intermediate precursors from all clones tested were assayed with whole cell lysate of aerobic and anerobic gut flora, all of the antibodies in all of the clones reacted with gut flora whole cell lysate. FIG. 9 shows a heat map of the 684-6B clone reacting at each mAb with aerobic whole cell lysate (WCL). Similar results were obtained with anerobic WCL. When analyses were performed to determine antigen drive mediated by gut flora, it was found that, indeed, there were increases in antibody affinity coincident with progressive somatic hypermutation in the AHI clones, though less so than for gp41.

[0125] Western blot of AHI gp41 mAbs with anerobic and aerobic gut flora whole cell lysates. Next, the reactivity of the inferred intermediate #2 in FIG. 6 (HV00276) was determined with both anerobic and aerobic WCL in blue native PAGE (FIGS. 10A and B) and in SDS-PAGE (FIGS. 11 and 12). In blue native gel analysis, the 684-6B clone mAb reacted with a 520,000 Da molecule in both aerobic and anerobic gut samples (FIGS. 10 A and 10B). Moreover, mAb 276 also reacted with the 480 KDa MW marker that is phycoerthryn (FIGS. 10A and 10B). FIGS. 11 and 12 show that under SDS-PAGE non-reducing (FIG. 11) and reducing (FIG. 12) conditions, strong bands are seen again at .about.520,000 Da. Also smaller band is seen at approx 60 and 50 Kd as well as in the native marker under reducing conditions (FIG. 12). The native marker is again phycoerythrin (PE) showing polyreactivity against PE by the 684-6B clone mabs.

[0126] Importantly, the somatically mutated original 2F5 and 4E10 broad neutralizing antibodies also reacted with protein bands in gut flora WCL with 2F5 reacting with .about.300,000 Da molecule and .about.80,000 Da molecules in aerobic WCL and 4E10 reacting with .about.80,000 and 100,000 Da molecules in aerobic WCL. In FIG. 12 (SDS-PAGE under reducing conditions), it is seen that HV00276 (intermediate 684-6 ab #2) binds to an .about.520,000 Da band in aerobic and anerobic WCL while 2F5 reacts with an .about.80,000 Da band and 4E10 with an approximately 60,000 da band in aerobic WCL.

[0127] It has been shown previously that the broad neutralizing antibodies 2F5, 4E10 and 1b12 are polyreactive antibodies that bind to multiple host antigens. Thus, the question is, if the initial response to HIV is by a polyreactive antibody response, why are not polyreactive antibodies made that broadly neutralize? Two possibilities have been considered.

[0128] First, it has been shown that the germline of 1b12, 2F5 and 2G12 do not bind to HIV gp120 or gp41 while the somatically hypermutated antibodies do bind (Xiao et al, Biochem. Biophys. Res. Commun. 390:404-9 (2009)). Thus, the notion is for many of the epitopes of broad neutralizing antibodies, the immunogens the field has been using do not target the B cell receptors of the naive B cells they are targeting. The germline of the 1b12 has now been studied for lipid reactivity and for gut flora whole cell lysate activity and it has been found that, indeed, the germline 1b12 reactivity is negative to HIV gp120 envelope while the reactivity of the somatically mutated 1b12 is very high to HIV gp120 (FIG. 13). In contrast, the reactivity of the germline of 1b12 is very high to cardiolipin while the somatically mutated polyreactive original 1b12 mAb reactivity to cardiolipin is very low though not negative (FIG. 13). Moreover, the germline of 1b12 is reactive as well with gut flora whole cell lysate, while the mature original somatically mutated 1B12 mAb is only weakly reactive (Table 2).

TABLE-US-00002 TABLE 2 Reactivity of Broadly Neutralizing Monoclonal Antibodies 2F5, 4E10, 1612, and 2G12 with Gut Flora and Their Germline Antibodies With Gut Flora Anerobic Gut Aerobic Gut MAb gp41 gp120 Flora WCL Flora WCL Reactivity in Luminex Units 1b12 original NA 5106 148 384 1b12 germline NA <80 524 1127 2F5 original 32717 9237 103 100 2F5 germline NA NA NA NA 4E10 original 4E10 germline NA NA NA NA 2612 original 2612 germline <80 <80 <80 <80 17b original 1433 <80 <80 <80 CCR5 binding site antibody

[0129] Second, it has been hypothesized that the polyreactivity of 2F5, 4E10 and 1b12 target the B cells making these types of antibodies for deletion or anergy (Haynes et al, Science 308:1906-8 (2005); Haynes et al, Human Antibodies 14:59-67 (2005); Alam et al, J. Immunol. 178:4424-35 (2007)). This hypothesis has recently been proven for the 2F5 VH in 2F5 FH homozygous knock-in mice (Verkoczy et al, Proc. Natl. Acad. Sci. USA 107:181-6 (2010)) and now in 4E10 VH homozygous mice (FIG. 14). In both animal models of knock-in of the broadly reactive somatically mutated VHs, the mutated VHs are sufficiently autoreactive to cause deletion in the bone marrow and to invoke multiple tolerance mechanisms in the periphery.

[0130] In summary, the results described above demonstrate:

[0131] The initial antibody response to HIV is focused on non-neutralizing Env gp41 epitopes.

[0132] The initial gp41 antibody response arises from preexisting somatically mutated, polyreactive "natural" antibody clones whose germline Ab do not react with gp41 but whose inferred intermediate Abs do react with gp41.

[0133] While the antibody members of gp41 antibody-reactive clones are polyreactive and cross-react with lipids and other self cellular antigens, the affinity of anti-gp41 antibodies increases as somatic hypermutation occurs, indicating gp41 antigen drive.

[0134] Initial HIV-induce clonal development however is not efficient nor high affinity--perhaps due to self mimicry, leading to a mixture of HIV Env-reactive and non-reactive antibody clone members.

[0135] The germline of broad neutralizing antibodies 1b12, 2F5 and 2G12 do not appear to react with their inferred germline antibodies.

[0136] IgM antibodies isolated from AHI or uninfected subjects that bind to gp41 also bind to gut flora whereas gp41 negative IgMs do not bind gut flora antigens

[0137] The germline of 1b12 reacted with lipids and gut flora, implying origin from pre-existing polyreactive natural antibody producing naive B cells that likely originated from B cell clones originally targeted against gut flora.

[0138] The somatically mutated 2F5, 4E10 and 1b12 broadly neutralizing antibodies all react with antigens in gut flora whole cell lysates, indicating that these antibodies likely derived from clones of naive B cells originally targeted to gut flora.

EXAMPLE 2

[0139] The enrichment and identification of a protein band in intestinal bacterial lysate reactive with mAb HV00276 is shown in FIG. 52. Western blot analysis following a Native PAGE gel run shows that mAb HV00276 binds to a .about.520 kDa protein band in an anaerobe and aerobe intestinal bacterial lysate. Protein fractions from the bacterial lysate having a molecular weight of .about.500 kDa were collected following size exclusion chromatography (SEC). SEC fractions show enrichment of the 520 kDa protein by Coomassie Blue (1), silver staining (2) and western blotting (3, arrow) with mAb HV00276. Isoelectric zoom fractionation shows migration of the mAb reactive protein to gel compartment A4 with pH6.2-7.

[0140] The 520 kDa band from the enriched fractions was subjected to LC-MS analysis for protein identification. RNA polymerase .beta., .beta.' and .alpha. subunits were identified (see FIG. 53).

[0141] E. coli RNA polymerase core protein and holoenzyme (core protein+ .sigma. subunit) (Epicentre Biotechnologies, Madison, Wis.) were run on a NativePAGE gel, and the reactivity of mAb HV00276 was detected using western blotting. Reactivity to both core and holoenzyme was detected indicating that mAb HV00276 binds to RNA polymerase core protein.

[0142] E. coli RNA polymerase core protein (Epicentre Biotechnologies, Madison, Wis.) was run on a denaturing SDS-PAGE gel under both reducing (Red) and non-reducing (NR) conditions (left panel). On denaturing SDS-PAGE, the individual subunits (.beta., .beta.', .alpha. and .omega.) of the core protein can be resolved and visualized following Coomassie Blue staining (right panel). Western blot analysis of the transferred gel shows that the 276 mAb binds only to the 37 kDa .alpha.-subunit of the RNA polymerase core protein. No reactivity of HV00503 mAb, which was negative for intestinal bacterial lysate proteins, was observed with any of the core protein subunits.

EXAMPLE 3

[0143] To understand how self tolerance may influence protective humoral responses to HIV-1, it is crucial to determine which self antigens are mimicked by HIV-1 epitopes and where/when these self antigens are exposed to T- and B lymphocytes. It is shown in FIG. 30 that monoclonal human antibodies specific for epitopes of the HIV-1 gp41 MPER also react with self-antigens present in acetone fixed mouse 3T3 cells. As shown in FIG. 31, at least four discrete molecules can be immunoprecipitated from mouse 3T3 cells by biotinylated 2F5 antibody. The dominant species precipitated has an apparent molecular mass of approximately 50-54 kDa.

[0144] A conserved mammalian protein, KYNU, carries the core 2F5 epitope and has a molecular mass of 51 kDa (FIG. 32). The 2F5 core epitope is present in the KYNU of many vertebrate species (FIG. 33) and is present in the conserved H3 domain of KYNU (FIG. 34). As shown in FIG. 35, the ELDKWA region (SEQ ID NO: 2) is in a well-ordered alpha helix. The DKW motif is not surface-exposed.

[0145] Binding of the 2F5 antibody to human KYNU may require a distortion of the H3 domain, potentially resulting in a slowed K.sub.on. As shown in FIG. 36, in H3, the D and W residues likely have exposed side chains but K is buried. The 2F5 antibody may necessarily "distort" the H3 helix to bind the ELDKWA epitope (SEQ ID NO: 2). Under physiological conditions, KYNU is thought to be a homodimer. The ELDKWA motif (SEQ ID NO: 2) may be available to KYNU monomers but is unlikely to be accessible when KYNU forms dimers (FIGS. 37 and 38).

[0146] Putative germline 2F5 antibodies also react with rhKYNU (FIGS. 39 and 40). This is an important point in that it demonstrates that KYNU could be the original ligand of B cells that eventually produced the mutated, high affinity 2F5 antibody. As shown in FIG. 41, the 2F5 antibody avidly reacts with a peptide (DP178-Q16L) containing the 2F5 epitope whereas anti-KYNU antibody does not (see also FIGS. 42 and 43).

[0147] 13H11, a non-neutralizing mouse HIV-1 MPER monoclonal antibody that recognizes an epitope proximal to the 2F5 determinant, does not bind rhKYNU (FIG. 44). FIG. 45 provides a mapping of residues that distinguish the binding sites of 2F5 and 13H11 monoclonal antibodies to the HIV-1 gp41 MPER. The data shown in FIG. 46 demonstrate competitive inhibition of 2F5 binding to rhKYNU by recombinant HIV-1 gp140 env (JRFL), DP178-Q16L, and an irrelevant peptide antigen, R4A.

[0148] JRFL recombinant HIV-1 gp140 comparably inhibits the binding of 2F5 to JRFL (homologous inhibition) and to rhKYNU (heterologous inhibition) (FIG. 47). The similarity of the inhibition curves indicates that a single, common epitope is responsible for 2F5 binding to both JRFL and rhKYNU.

[0149] As shown in FIG. 48, 2F5 monoclonal antibody binds both plate-bound and soluble rhKYNU comparably. Surface plasmon resonance studies demonstrate that both 2F5 and its unmutated precursors are capable of binding avidly to rhKYNU (FIG. 49). The slower K.sub.on is consistent with the 2F5 antibodies distorting the native KYNU structure in order to achieve maximal interaction. K.sub.off rates are very slow indicating that the bound KYNU interacts stably with all 2F5 types.

[0150] SPR binding analysis shows that the 2F5 mAb and its RUA (2F5-GL1 and 2F5-GL3) bind to KYNU (FIG. 50). Each of the antibodies was captured on a human anti-Fc immobilized sensor surface and soluble KYNY was injected at concentrations 50, 30, 20, and 10 .mu.g/mL. Overlay of the binding curves show specific binding of KYNU to each antibody. Non-specific binding was measured using a control mAb (Synagis, anti-RSV) which showed no binding to KYNU.

EXAMPLE 4

[0151] To determine whether 2F5 reactivity to fixed 3T3 cells could be inhibited by proteins/polypeptides containing the 2F5 MPER core epitope (ELDKWA) (SEQ ID NO: 2), 2F5 monoclonal (10 .mu.g/ml) antibody was reacted with increasing molar concentrations of homologous (JRFL and DP178) or heterologous (R4A) inhibitors (1 hr, 25.degree. C.). These mixtures were subsequently added to hydrated/blocked slides covered with methanol/acetone fixed 3T3 cells for (2 hr, 25 .degree. C.). Slides were rinsed and then washed overnight in 250 ml (PBS with 0.1% Tween-20 and 0.5% BSA). Washed slides were overlayed with goat anti-human IgG-FITC (1:400 in PBS with 0.1% Tween-20 and 0.5% BSA). After 1 hr., slides were washed, coversliped in Fluoromount-G. Twenty-four hr. later, fluorescence images were acquired using a Zeiss Axiovert 200M confocal microscope at 200.times. magnification and a fixed 300 msec exposure time.

[0152] Homologous inhibitors, the JRFL protein and, to a lesser extent, DP178 polypeptide, inhibited 2F5 binding to 3T3 cells. An irrelevant polypeptide, R4A, showed no inhibition. (See FIG. 51.) These data demonstrate that a substantial amount of 2F5 reactivity to fixed 3T3 cells is determined by protein-protein interaction rather than un-specific lipid binding. Thus, proteins, like KYNU, may be primary autoligands for 2F5.

EXAMPLE 5

[0153] As described above, the present invention relates to a vaccine strategy that comprises administering HIV envelope proteins (peptides or polypeptides) to, first, target B cells that express unmutated ancestor antibodies that are able to give rise to broadly neutralizing matured antibodies and, then, drive maturation of the B cell clones toward the desired breadth of neutralization by boosting the B cells that are undergoing somatic maturation with selected HIV envelope proteins (peptides or polypeptides). The development of the strategy involved reconstruction of this maturation pathway. Desired final (mature) antibodies were isolated from a patient who produces broadly neutralizing antibodies and the antibodies were characterized. The respective putative ancestral antibodies were inferred and expressed as real antibodies and a determination was made as to what they bind. The notion is that the B cells expressing unmutated "ancestral" and intermediate antibodies will affinity mature when triggered with the appropriate proteins (peptides or polypeptides) to yield the broadly neutralizing antibody-secreting B cells observed in the patient.

Selection and Isolation of Cross-Clade Neutralizing Monoclonal Antibodies CH01, CH02, CH03, CH04 and CH05

[0154] Approximately 30,000 memory B cells obtained from frozen PBMCs of subject 707-01-021-9 were screened and 28 cultures were found that neutralized >50% of CAP45 infectivity (FIG. 56). Monoclonal antibodies CH01, CH02, CH03, CH04 and CH05 (CH01-CH05) were isolated from four of these culture wells (1-27-G2, 1-27-G11, 1-19-F10 and 1-19-B7) (FIG. 56).

[0155] Amplification and sequencing were carried out of the V-heavy and V-light chains obtained from the RNA-later-treated memory B cells frozen at the time of screening. Cultures 1-27-G2 and 1-19-F10 contained only one pair (3.about.20/.kappa.3.about.20; CH01 and CH02 monoclonal antibodies, respectively), which indicates that the cultures were monoclonal and that the CH01 and CH02 are natural antibodies. Conversely, 1-27-G11 and 1-19-B7 contained multiple V-heavy and V-light chains, indicating that the cultures were oligoclonal.

[0156] To identify the natural pairs from these latter cultures, single-cell sorted memory B cells, collected at the time of initial screening, were amplified and sequenced. CH03 and CH04 (both 3.about.20/.kappa.3.about.20) were natural pairs isolated from cultures 1-27-G11 and 1-19-B7, respectively.

[0157] Human B-cell hybridomas were generated from culture 1-19-B7 by further expanding and cloning by sequential limiting dilutions the memory B cells for approximately 4 weeks. By this means, the CH04 natural antibody was obtained and CH05 was identified, which was produced by a lesser population of expanded memory B cells and expressed the same 3.about.20 V-heavy of CH04 but paired with a different .kappa.1.about.6 V-light chain.

[0158] The CH01-CH03 monoclonal antibodies were obtained by transfecting the V-heavy and V-light pairs into 293T cells and expressed in an IgG1 backbone as previously described (Liao et al, J Virol Methods. 158(1-2):171-9 (2009)). Monoclonal antibodies CH04 and CH05 were instead purified from the hybridoma B cell lines.

[0159] These data demonstrate that the strategy allows quick identification of neutralizing monoclonal antibodies in approximately 2 weeks and production of natural monoclonal antibodies as early as one month. Furthermore, this method resolves the uncertainties of the classic phage display libraries related to the precise characterization of a monoclonal antibody being true to the natural antibodies that are represented in the in vivo repertoire. Finally, reported for the first time is the production of two natural human B-cell hybridomas that broadly neutralize HIV-1.

Genomic Characterization of the CH01-CH05 Antibodies

[0160] It was determined that the CH01-CH05 antibodies are all member of the same clonal family based on the following factors: (1) V(D)J families; (2) length of the HCDR3; (3) nucleotide sequences of the HCDR3 region and of the n-insertions.

[0161] The analysis of the heavy chains showed that CH01-CH05 are IgG1 antibodies, sharing the same V 3.about.20*1/J 2*01 rearrangement (Table 3). They also share the same D region which resulted from the D-D fusion of the 3.about.10*1 and the 2OF15*2/inv regions (Table 3). The HCDR3 is 26 amino acids long (Table 3). N-insertions were also of the same length and shared a nucleotide makeup compatible with the notion that CH01-CH05 monoclonal antibodies are clonally related (FIG. 57A). The V-heavy sequences of CH04 and CH05 are identical (FIG. 57A), which suggests that the moment in which the V-light chain peripheral editing occurred was intercepted.

TABLE-US-00003 TABLE 3 Main characteristics of the CH01-CH05 VH and VL sequences V-heavy chain V-light chain HCDR3 Mutation LCDR3 Mutation V D J length rate* Isotype V J k/l length rate CH01 3~20 3~3, 2OF15/inv 2 26 0.120 IgG1 3~20 1 k 9 0.091 CH02 3~20 3~3, 2OF15/inv 2 26 0.118 IgG1 3~20 1 k 9 0.116 CH03 3~20 3~3, 2OF15/inv 2 26 0.152 IgG1 3~20 1 k 9 0.138 CH04 3~20 3~3, 2OF15/inv 2 26 0.153 IgG1 3~20 1 k 9 0.110 CH05 3~20 3~3, 2OF15/inv 2 26 0.153 IgG1 1~6 2 k 9 -- *Mutation rates are calculated from putative reverted unmutated ancestor variable heavy and variable light chains inferred from the sequences of each individual monoclonal antibody independently.

[0162] Seemingly to the V-heavy chains, CH01-CH04 shared the same VL.kappa.3.about.20/JL.kappa.1 rearrangement (FIG. 57B), an LCDR3 of the same length (9 aminoacids) and similar n-insertions (FIG. 57B). The V-light chain of monoclonal antibody CH05 was instead unrelated (FIG. 57C), with a different VL.kappa.1/JL.kappa.2 rearrangement, LCDR3 length and n-insertions. It is contemplated that the biology underlying the pairing of the V-light chains to the VH3.about.20 chain is that the VH3.about.20/VL.kappa.3.about.20 chain pairs (CH01-CH04) preceded the VH3.about.20/VL.kappa.1.about.6 pairing (CH05) because higher VL.kappa. numbers are closer to the J.kappa. locus and, therefore, ancestor antibodies would have had to rearrange VL.kappa.3 first and then VL.kappa.1. Furthermore, the low-numbered J.kappa. loci have to come before the high-numbered. Therefore, the transition from VL.kappa.3/JL.kappa.1 to VL.kappa.1/JL.kappa.2 is consistent with simple editing. Finally, the phylogenetic tree shown in FIG. 58, and discussed below, provides further very strong evidence that the VL.kappa.3/JL.kappa.2 rearrangement happened first.

[0163] Next, a determination was made of the genetic relationship of the CH01-CH05 monoclonal antibodies by constructing the phylogenetic tree of the V-heavy chains (FIG. 58). To do so, the putative reverted unmutated ancestors of the CH01-CH05 antibodies were inferred by applying the maximum likelihood analysis on the observed antibodies as a whole. Using this method, two possible RUAs (0219-RUA1 and 0219-RUA2) were predicted that differed only for a single silent nucleotide substitution (G or T) in position 329 (FIG. 59). The putative RUAs were also predicted by analyzing each observed monoclonal antibody independently. With this method, 9 RUA antibody candidates were identified: one for CH01 (CH01-RUA1), two for CH02 (CH02-RUA1 and CH02-RUA2), four for CH03 (CH03-RUA1, CH03-RUA2, CH03-RUA3 and CH03-RUA4) and two for CH04 (CH04-RUA1 and CH04-RUA2). The alignment of all the computed putative RUAs is shown in FIG. 59.

[0164] The phylogenetic tree of the V-heavy chains (FIG. 58) shows that CH02 and CH03 are genetically close to each other and that CH03 is the most somatically mutated monoclonal antibody of the family.

[0165] Taken together, these data demonstrate that CH01-CH05 are clonally-related heavily somatically mutated monoclonal antibodies that share a long HCDR3 and harbor a D-D fusion rearrangement. Moreover, this is the first description of peripheral light chain editing in humans.

CH01-CH05 Monoclonal Antibodies Broadly Neutralize Tier 2 HIV-1 Isolates and Bind to a Limited Set of Monomeric gp120/gp140 HIV-1 Envelope Proteins.

[0166] The neutralization breadth of the CH01-CH05 antibodies was tested against a panel of 96 HIV-1 primary isolates. The panel comprised 4 tier 1A isolates, 3 tier 1B isolates (2 clade B and 1 clade AE) and 89 tier 2 isolates which included 10 clade A, 21 clade B, 27 clade C, 4 clade D, 7 clade G, 1 clade AE, 1 clade AD, 9 CRF01_AE and 9 CRF02_AG viruses.

[0167] As predicted by the genetic analysis, CH01-CH05 shared a very similar pattern of neutralization (Table 4). All the antibodies neutralized viruses from multiple clades and the breadth of neutralization ranged from 44.9% (43/96 isolates) of CH01 to 34.7% (33/95 isolates) of CH02. CH03, CH04 and CH05 neutralized 43.2% (41/95), 43.2% (41/95) and 44.2% (42/95) isolates, respectively. None of the antibodies neutralized tier 1A isolates. Tier 1B isolates were neutralized only by CH01 (2 out of 3), CH02 and CH03 (1 out of 3) but not by CH04 or CH05. Conversely, CH01-CH05 showed larger breadth of neutralization against tier 2 viruses. CH01 preferentially neutralized CRF02_AG isolates (7/9; 77.8%), followed by clade A (7/10; 70%), CRF01_AE (5/9; 55.6%), clade B (9/21; 42.9%), clade C (11/27; 40.7%), and clade G (1/7; 14.3%) isolates. Clade D viruses were not neutralized. Conversely, it is important to note that the CH01-CH05 monoclonal antibodies strongly neutralized AE.CM244.ec1 (Table 4). The preferential neutralization of tier 2 viruses over tier 1 viruses is important in that previous work demonstrated that broad neutralization of easy-to-neutralize tier 1 isolates does not translate into breadth against more difficult-to-neutralize tier 2 isolates and, therefore, those kinds of antibodies could be of limited help in preventing or controlling HIV-1 infection.

TABLE-US-00004 Neutralization profile of CH01-CH05 monoclonal antibodies ##STR00001## ##STR00002## ##STR00003## ##STR00004##

[0168] In comparison, the recently described PG9 and PG16 quaternary antibodies, shown in the table, neutralized 73/83 (88%) and 69/83 (83.1%) tier 2 isolates, respectively. Interestingly, with only one exception (T251-18), PG16 neutralizes a subset of the isolates neutralized by PG 9 and the CH01-CH05 broadly neutralizing antibodies neutralize a subset of viruses neutralized by PG16. This finding is compatible with the hypothesis that the CH01-CH05 epitope is related to that of PG9/PG16.

[0169] Next, the potency of the CH01-CH05 antibodies against the neutralization-sensitive isolates was evaluated. Overall, the median IC50 was approximately 1 .mu.g/ml with an average IC50 ranging from 2.4 to 5.6 .mu.g/ml. CH03 showed the strongest potency among the CH01-CH05 antibodies with a mean IC50 of 2.4 .mu.g/ml and a median IC50 of 0.46 .mu.g/ml, comparable to those of PG9 (mean IC50=2.1 .mu.g/ml; median IC50=0.11 .mu.g/ml) but weaker than those of PG16 (mean=0.67 .mu.g/ml; median<0.02 .mu.g/ml). CH01, the broadest neutralizer, showed a mean and median IC50s of 3.7 and 1.1 .mu.g/ml, respectively. CH02, CH04 and CH05 mean IC50s were 4.9, 4.7 and 4.3 .mu.g/ml, and median IC50s were 0.97, 0.8 and 0.79 .mu.g/ml, respectively.

[0170] The ability to neutralize transmitted founder viruses is another critical parameter to evaluate. As shown in Table 4, CH01-CH05 bNabs were able to neutralize 3/3 (100%) clade A, 2/9 (22.2%) clade B and 2/3 (66.7%) clade C transmitted founder viruses.

[0171] Taken together, these data indicate that the clonal family of CH01-CH05 antibodies broadly neutralize tier 2 isolate from multiple clades, including transmitted founder viruses. This is the first report of a clonal family of broadly neutralizing antibodies. Since there was no significant differences in the pattern of neutralization of CH05 compared to that of the other broadly neutralizing antibodies of the clonal family, these results also indicate that the edited VL.kappa.1.about.6 chain permitted the neutralization of the tested isolates at comparable levels to the VL.kappa.3.about.20 chain.

[0172] In contrast to the mature antibodies, the inferred putative RUAs did not show such breadth of neutralization. Yet, few isolates were potently neutralized. The neutralization profile of 6 inferred RUAs tested on a panel of 24 isolates is shown in Table 5. It is important to note that CH03-RUA1, CH03-RUA4 and CH03-RUA3 neutralized AE. CM244.ec1 isolate with IC50 of 4.45, 5.26 and 18.8 .mu.g/ml, respectively. Also, B.WITO4160.33 was potently neutralized by all the RUAs tested (IC50s from 0.06 to 0.47 .mu.g/ml). A.Q23.17 isolate was also neutralized very potently by CH01-RUA1, CH03-RUA1, CH03-RUA3 and CH03-RUA4 with IC50s<0.02 .mu.g/ml. Conversely, CH02-RUA1 and CH03-RUA2 neutralized A.Q23.17 at IC50s three orders of magnitude higher, showing the same pattern of neutralization of C.ZM233M.PB6.

TABLE-US-00005 ##STR00005## ##STR00006##

Binding of CH01-CH05 Antibodies to Monomeric gp120/gp140 HIV-1 Envelopes.

[0173] To determine which monomeric envelope could be used in a vaccine formulation to bind to B cells and trigger the production of CH01-CH05-like antibodies, CH01-CH05 monoclonal antibodies and RUAs were tested for binding to a panel of 32 monomeric envelopes. Table 6 shows the EC50s expressed in .mu.M.

TABLE-US-00006 TABLE 6 CH01-CH05 ELISA binding to monomeric gp120/gp140 envelope proteins CH01- CH03- CH02- CH02- Source Clade Env Env Name CH01 CH02 CH03 CH04 CH05 RUA1 RUA1 RUA1 RUA2 synagis Chronic A gp140 00M SA 4076 NB NB NB NB NB NB NB NB NB NB Chronic A gp140 VRC A NB NB NB NB NB NB NB NB NB NB Chronic Anc gp140 US-1* NB NB NB NB NB NB NB NB NB NB Chronic B gp140 VRC B NB NB NB NB NB NB NB NB NB NB Chronic B gp140 JRFL NB NB NB NB NB NB NB NB NB NB Chronic C gp140 97CNGX2F 140 CF NB NB NB NB NB NB NB NB NB NB Chronic C gp140 DU 123 NB NB NB NB NB NB NB NB NB NB Chronic C gp140 CN54 NB NB NB NB NB NB NB NB NB NB Chronic G gp140 HV 14000 (DRCBL) NB NB NB NB NB NB NB NB NB NB Chronic B gp120 W61D NB NB NB NB NB NB NB NB NB NB Chronic B gp120 MN NB NB NB NB NB NB NB NB NB NB Chronic B gp120 VBD2** NB NB NB NB NB NB NB NB NB NB Chronic E gp120 A244gD+ 7.8 150 34.5 23.1 28.7 >666.7 >666.7 NB NB NB Chronic C gp120 ZM651 NB NB NB NB NB NB NB NB NB NB Chronic AE gp120 CM 243 12.7 >666.7 97.3 >666.7 >666.7 NB NB NB NB NB Consensus A1.CON gp140 A1.con.env03 140 CF NB NB NB NB NB NB NB NB NB NB Consensus AE.CON gp140 HV 13700 NB NB NB NB NB NB NB NB NB NB (AE.con.env03 140 CF) Consensus B.CON gp140 B.con.env03 140 CF NB NB NB NB NB NB NB NB NB NB Consensus C.CON gp140 C.con.env03 140 CF NB NB NB NB NB NB NB NB NB NB Consensus M gp140 Con 6 140 CF NB NB NB NB NB NB NB NB NB NB Consensus M gp140 Con S 140 CFI NB NB NB NB NB NB NB NB NB NB T/F A gp140 HV13341 (0219) NB NB NB NB NB NB NB NB NB T/F B gp140 FIKE gp140C NB NB NB NB NB NB NB NB NB NB T/F B gp140 HV00043 NB NB NB NB NB NB NB NB NB NB (63521 TC21 140C) T/F B gp140 HV00044 NB NB NB NB NB NB NB NB NB NB (6240 TZ5 140C) T/F B gp140 HV00045 NB NB NB NB NB NB NB NB NB NB (6235714 D3 140C) T/F B gp140 HV00046 63.2 240 NB NB NB NB NB NB NB NB (902114 B2 140C) T/F B gp140 HV00049 NB NB NB NB NB NB NB NB NB NB (700010040 C9 140C) T/F B gp140 MOJO gp140C NB NB NB NB NB NB NB NB NB NB T/F C gp140 HV00047 NB NB NB NB NB NB NB NB NB NB (089C 140C.) T/F C gp140 HV00048 NB NB NB NB NB NB NB NB NB NB (1086C 140C) T/F B gp120 FIKE gp120 NB NB NB NB NB NB NB NB NB NB *SIV gp140 **gp120 no MPER

[0174] Binding to monomeric envelope was weak with the exception of gp120 A244gD.sup.+, which was bound by the CH01-CH05 antibodies with EC50s ranging from 7.8 .mu.M (CH01) to 150 .mu.M (CH02). In addition, and of extreme relevance for the selective targeting of precursors of B cells capable of secreting broadly neutralizing antibodies, also two putative RUAs showed some binding (Table 6). The other HIV-1 envelope that was bound by all the five mature antibodies was gp120 CM243, even though the mean EC50 was higher. The sequence of the A244 (CM244) Envelope is from McCutchan et al (AIDS Res. Hum. Retrovir. 8(11):1887-1895 (1992)) with the exception of aa substitutions of L124P, N196S, K198E, A212P and D284 N. In addition, there is a 30AA sequence from the gD protein of herpes simplex virus KYALVDASLKMADPNRFRGKDLPVLDQ (SEQ ID NO: 7) at the N-terminus of gp120 A144 (CM244). This sequences comprises the receptor binding sites of the gD protein required for HSV entry and infection (Yoon et al, J. Virol. 77:9221 (2003), Connolly et al, J. Virol. 79:1282-1295 (2005), Campadelli-Fiume et al, Rev. Med. Virol. 17: 313-326 (2007)). In the RV144 Thai vaccine trial where this A244 gp120 was used as an immunogen, the subjects responded to the gD protein in both the MN gp120 and the A244 gp120 with both IgA (FIG. 60) and IgG (FIG. 61) gD antibodies. FIG. 62 shows that there are two potential sites of interest in the gD peptide that may mimic the alpha 4 beta 7 binding site of gp120 LPV and LDQ. Thus this raises three possibilities:

[0175] 1. Motif for gp120 binding to a4b7 is LDV and LDI

[0176] HSV gD LPV and LDQ

This raises the question whether antibodies to gD can block binding of HIV gp120 to a4b7.

[0177] 2. LDQ of HSV-gD is a receptor binding site for host cellular receptor heparan sulfate (Yoon et al, J. Virol. 77:9221 (2003)). This raises the question whether antibodies to gD can block binding HIV Env to heparan sulfate.

[0178] 3. The LDQ is also the receptor binding site for the second HSV receptor HVEM. The anti-HSV antibody response to LDQ could be protective against HSV (Yoon et al, J. Virol. 77:9221 (2003)). Therefore, an anti-gD response could be protective for HIV by reducing active infection.

[0179] Lack of binding to most monomeric gp120/gp140 envelopes indicates that CH01-CH05 bind to a conformation-sensitive, quaternary antibody, preferentially expressed on trimeric envelopes. Similar findings have been reported for PG9 and PG16 antibodies (Walker et al, Science 326(5950):285-9 (2009)). Conversely, the strong binding to the A244gD.sup.+ gp120 envelopes strongly suggested that the co-expression of the HSV-1 glycoprotein D restored the functional epitope.

[0180] To investigate the role of HSV-1 glycoprotein D in enhancing the binding of the CH01-CH05 antibodies and to detect binding of the RUAs to envelopes at levels that can be below the threshold of detection of standard ELISAs but still physiologically relevant, the constant of dissociation (k.sub.d) of the CH01-CH05 antibodies and RUAs to A244gD.sup.+ and A244gD.sup.- gp120 envelopes was measured using surface plasmon resonance (Table 7). A244gD.sup.+ consistently showed a k.sub.d at least an order of magnitude lower than A244gD.sup.- but, even more importantly, all the RUAs bound to A244gD.sup.+ gp120 with k.sub.d's ranging from 790 nM to 26.7 nM. The surface plasmon reasonance patterns for these data are shown in FIGS. 63-66. Also seen in FIG. 66 is that the transmitted founder virus 6240 bound with sub-nanomolar Kd to PG9. Taken together these data demonstrate that the A244 gD+ envelope as well as the 6240 transmitted founder envelope were in a similar conformation as the gp120 found in the native Env trimer, and thus should be in the correct confirmations for use as immunogens.

TABLE-US-00007 TABLE 7 Constant of dissociation of the CH01-CH05 monoclonal antibodies and RUAs detected by surface plasmon resonance Constant of dissociation K.sub.d (nM) Env Clade CH01 CH02 CH03 CH04 CH05 PG9 PG16 CH01-RUA1 CH02-RUA1 CH02-RUA2 CH03-RUA1 A244 gD.sup.+ gp120 E 8.8 450 15.6 25.5 26.1 4.8 450 26.7 790 410 83.5 A244 gD.sup.- gp120 E 340 N/A 728 410 340 N/A N/A 2460 N/A N/A 270 63521 TC21 gp140 B (T/F) N/A N/A N/A N/A N/A 0.75 160 N/A N/A N/A N/A 624008 TA5 gp140 B (T/F) 300 N/A 1060 360 1450 210 110 N/A N/A N/A N/A

Autoreactivity and Polyreactivity Profile of CH01-CH05 Broadly Neutralizing Antibodies.

[0181] Table 8 shows that CH03 is autoreactive with RNP, histone and centromere B autoantigens. Presence of antibodies binding to centromere in CH03 was also found using indirect fluorescent antibody staining on HEp-2 cells (FIG. 67). Table 12 reports the binding (measured by Luminex assay) of CH01-CH05 to 4 non-HIV antigens. The data show that CH01-CH-3 are strongly polyreactive. CH04 and CH05 polyreactivity is still detectable even through at a much lower level. Conversely, PG9 and PG16 showed no polyreactive abilities. These data point out a potentially relevant difference on the biology of the respective developments between the CH01-CH05 and PG9 and PG16 antibodies.

TABLE-US-00008 TABLE 8 Autoreactivity (Athena) Criteria for positive: >50 Conc. ug/ml SSA SSB Sm RNP Scl 70 Jo 1 dsDNA Cent B Histone Neg Control -- -- -- -- -- -- -- -- -- -- Pos Control 1 -- 397 Pos Control 2 -- 631 699 1073 441 Pos Control 3 -- 544 458 402 575 4E10 50 306 254 9 20 3 156 19 31 333 25 247 206 7 15 4 138 8 19 274 12.5 169 124 5 9 3 87 6 13 160 6.25 115 93 4 6 2 65 3 9 113 CH01 50 8 5.5 4 8 4 3.5 32 22 26 25 6.5 5.5 4 5.5 3 2.5 17 14 17.5 12.5 5 5 3 4.5 2 2 9.5 10 13.5 6.25 5.5 5.5 2.5 4 1.5 2 6.5 7 10 CH02 50 5.5 4.5 3.5 12.5 2.5 2 16 12.5 15.5 25 6 4.5 3 9 2 1.5 10 9.5 11.5 12.5 5 3.5 2.5 6 1.5 1 5 7 8.5 6.25 5 5 2.5 5 2 1.5 3 6 7 CH03 50 9 7 24 132 12 5 0 98 844 25 6 4 10 70 7 4 38 34 386 12.5 6 6 9 74 8 4 0 30 359 6.25 5 5 7 51 4 3 0 19 231

TABLE-US-00009 TABLE 12 Evaluation of the polyreactivity of the CH01-CH05 antibodies measured by Luminex assay. Anaerobic Aerobic Gut Influenza HA HCV E2 Gut Flora Flora (Wisconsin) CH01 17.5 196.7 19.2 0 CH02 333.3 258.8 28.5 0.3 CH03 2909.5 286.3 97.5 1 CH04 0.5 17 3.5 2.5 CH05 1.3 23.5 9 5.5 PG9 0 0 0 0 PG16 0 0 0 0 4E10 89.2 83 96.2 0 Synegis 0.5 2.5 1.5 1

Results are expressed as background substracted RFUs using an antibody concentration of 50 .mu.g/ml Characterization of the Epitope Targeted by the CH01-CH05 Broadly Neutralizing Antibodies. PG16-Like Phenotype

[0182] It was determined that the CH01-CH05 antibodies share unique characteristics with the quaternary broadly neutralizing PG9 and PG16 antibodies recently described by Walker et al (PLoS Pathog. 6(8).pii:e1001028 (Aug. 5, 2010)). In particular, the CH01-CH05 bNabs were characterized as "PG-like" antibodies based on the following four criteria: (1) the point mutation of the asparagine into a lysine at position 160 (N160K) of the gp120 protein abrogates the neutralization of an otherwise neutralization-sensitive isolate, (2) neutralization of otherwise neutralization-sensitive isolates is abrogated when the virus is partially deglycosilated due to its production in cells treated with the mannosidase I-inhibitor kifunensine, (3) the epitopes are preferentially displayed in the context of envelope trimers but are not found on monomeric gp120 or gp140 envelopes, and (4) threading shows a high similarity with PG9 or PG16 bNAbs. As a representative of the CH01-CH05 clonal family of bNabs, CH01 was tested to determine if it met all the four criteria.

[0183] Table 9 shows the effect of the N160K point mutation on the CH01 neutralizing activity (IC50 and IC80) compared to that of PG9 and PG16 against a panel of wild-type and mutated isolates: clade A Q23.17 and clade B JR-CSF JRFL and 7165.18 isolates. CH01, PG9 and PG16 all strongly neutralize the wild-type Q23.17 (IC50s=0.014, 0.002 and 0.001 .mu.g/ml, respectively) and JR-CSF (IC50s=0.07, 0.003 and 0.003 .mu.g/ml, respectively) isolates. The introduction of the N160K mutation in the gp120 protein of Q23.17 and JR-CSF equally leads to complete abrogation of neutralizing activity by the three antibodies (IC50>50 .mu.g/ml). CH01, PG9 and PG16 also share the same neutralization pattern against JRFL and its mutants. Neither of them neutralizes wild-type JRFL. A single mutation at position 168 (E168K) reconstitutes a properly conformed epitope and results in potent neutralization (IC50s=0.044, 0.008 and 0.003 .mu.g/ml for CH01, PG9 and PG16, respectively) but the subsequent introduction of the N160K mutation reverts the effect of the E168K mutation, making the JRFL/E168K/N160K isolate neutralization resistant (IC50>50 .mu.g/ml) to all the three bNabs. Finally, 7165.18 is neutralized by CH01 (IC50=5.82 .mu.g/ml) and PG16 (IC50=11.8 .mu.g/ml) but not PG9 (IC50>50 .mu.g/ml) and, again, the N160K mutation abrogates neutralization by both CH01 and PG16. Taken together, these data indicate that the neutralization activity of CH01 is similarly affected by the signature N160K mutation in gp120 as PG9 and PG16.

TABLE-US-00010 TABLE 9 Effect of point mutations on sensitive glycosilation sites for PG9/PG16-like antibodies IC50 ug/ml IC80 ug/ml Clade Virus PG9 PG16 27G2 PG9 PG16 27G2 A Q23.17 0.002 0.001 0.014 0.005 0.003 0.035 Q23.17.N160K >50 >50 >50 >50 >50 >50 B JRCSF 0.003 0.003 0.070 0.008 0.012 >50* JRCSF.N160K >50 >50 >50 >50 >50 >50 JRFL >50 >50 >50 >50 >50 >50 JRFL.E168K 0.008 0.003 0.044 0.055 0.015 0.382 JRFL.N160K.E168K >50 >50 >50 >50 >50 >50 7165.18 >50 11.8** 5.82** >50 >50** >50** 7165.18.N160K >50 >50 >50 >50 >50 >50 *curve reached plateau at 78%. **curve reached plateau at 50-55%.

[0184] Another characteristic of PG9 and PG16 is that otherwise neutralization-sensitive viruses become resistant when 293T cells used to produce the virus are treated with kifunensine. FIG. 68 shows that CH01 neutralization of YU2 produced in293T cells is seemingly negated by treatment with 50 .mu.M of kifunensine.

[0185] Broadly neutralizing antibodies with a limited breadth of binding to monomeric gp120 and gp140 envelopes described above is typical of quaternary antibodies, whose epitope is correctly exposed in the context of the trimeric envelope.

[0186] Superimposition of CH01 onto threads of 7 distinct monoclonal antibodies showed that the structure of PG16 was the best fit to predict the 3D conformation of HC01 (Table 10). FIG. 69 shows the superimposition of CH01 onto the PG16 thread. PG9 and PG16 are characterized by a unique shape of the HCDR3 region that protrudes from the tip of the antibody structure in a "hammer-like" shape (Pancera et al, J. Virol. 84(16):8098-110 (2010)). No other antibody had been previously described with such characteristics. Notably, CH01 structure is very similar and the "head" of the "hammer" superimposes well with that of PG16 (FIG. 69). Being the HCDR3 shorter than PG9 and PG16, the sequence differs in some parts and this might be the structural explanation of the different breadth of reactivities between the CH01-CH05 antibodies and PG9/PG16.

TABLE-US-00011 TABLE Threading of 9 antibody sequences onto 7 antibody structures with the resulting models evaluated by normalized DFIRE score..sup.c Sequences Structures PG16.sup.a 47e.sup.a 412d.sup.a 17b.sup.a 48d.sup.a x5.sup.a e51.sup.a 27G2.sup.a PG9.sup.a PG16.sup.b 1.0 2.0 2.2 1.2 1.0 47e.sup.b 1.0 2.0 1.0 1.4 3.8 412d.sup.b 3.6 1.0 1.3 2.8 2.4 2.5 3.5 17b.sup.b 5.0 2.0 1.0 2.5 3.0 3.3 2.6 3.2 48d.sup.b 4.4 2.9 4.0 2.7 1.0 3.0 4.5 4.6 3.2 x5.sup.b 4.1 2.7 2.5 1.0 3.5 4.0 e51.sup.b 3.1 2.0 2.1 1.4 2.1 1.0 2.3 4.0 .sup.aAntibody sequences to be threaded, including PG16, 47e, 412d, 17b, 48d, x5, e51, 27G2 and PG9. .sup.bAntibody structures used as template, including PG16, 47e, 412d, 17b, 48d, x5 and e51. .sup.cAfter threading the variable region sequences of both heavy chain and light chain, the resulting model was evaluated using a normalized statistical potential (DFIRE). The smaller the score is, the better the sequence fits the template structure. Values are normalized: the Dfire score obtained after threading the sequences onto the structures are divided by the Dfire score of sequence threaded onto the matched structure (i.e PG16 sequence onto PG16 structure). 1. to 1.4 values are colored in green as they will probably be correct. 1.5 to 1.9 values are colored in orange 2.0 and above are colored in red as they are unlikely to be correct.

An interesting feature of quarternary antibodies is that they may be tyrosine sulfated in the same way as the CD4i antibodies (Huang et al, PNAS 101(9):2706-2711 (2004) Epub 2004 Feb. 23 and Pejchal et al, PNAS 107(25):11483-8 (2010)). Sequence analysis of CH01 performed with "sulfinator", a tyrosine sulfation prediction program, predicted one tyrosine that is likely to be sulfated (ARGTDYTIDDAGIHYQGSGTFWYFDL) (SEQ ID NO: 8) (Table 11). (Note that CH01 is called 1-27-G2.). Table 11 discloses SEQ ID NOS 81-85, 85-87, 86, 88-94, 94-95, 95-96 and 96, respectively, in order of appearance.

TABLE-US-00012 TABLE Tyrosine sulfation prediction for 1-27-G2, PG9, PG16 and CD4i antibodies. Heavy variable sequence CDR H3 sequence Sulfinator.sup.a Sulfosite.sup.b Sulfinator.sup.a Antibody Sequence E-value.sup.c Sequence SVM.sup.d Sequence E-value.sup.c 1-27-G2 none RGTDYTIDD 0.86 TDYTID 33 PG9 DYRNGYNYYDF 45 AFIKYDGSE 0.5 none YYDFYDGYY 0.5 PG16 none none none 47e none EDGDYLSDP 0.85 DGDYLSDPFY 7.8 DGDYLSDPFYYNHGMDV 38 412d PYPNDYDYAPE 24 NDYNDYAPEE 4.2 NDYNDYAP 14 DYNDVAPEE 0.59 DYAPEEG 40 17b none none none 48d none none none X5 none none none 23e none none none e51 none AAGDYADYD 0.69 none DYADYDGGY 0.95 YDGGYYYDM 0.54 CDR H3 sequence Sulfosite.sup.b Antibody Sequence SVM.sup.d Experimental Data 1-27-G2 none PG9 YYDFYDGYY 0.5 2 Tyr sulfated 10-fold down neutralization Pejchal et al, PNAS, 2010 PG16 none 1 Tyr sulfated 10-fold down neutralization Pejchal et al, PNAS, 2010 47e EDGDYLSDP 0.85 1 Tyr sulfated Role in binding to gp120 Huang CC et al, PNAS, 2004 412d 2 Tyr sulfated same Role in binding to gp120 Huang CC et al, PNAS, 2004 Choe, H et al, Cell, 2003 17b none 48d none X5 none Sulfated but no impact on binding Huang CC et al, PNAS, 2004 23e none e51 AAGDYADYD 0.69 3 Tyr sulfated DYADYDGGY 0.95 Loss in binding YDGGYYYDM 0.54 Huang CC et al, PNAS, 2004 Choe, H et al, Cell, 2003 .sup.aSulfinator: http://ca.expacy.org/tools/sulfinator/ .sup.bSulfosite: http://sulfosite.mbc.nctu.edu.tw/ .sup.cstatistical value of the match (smaller number are best) .sup.dSVM: support vector machine

Taken together these data strongly support the notion that CH01-CH05 bNabs are PG-like antibodies that recognize a quaternary epitope involving the V2 region of gp120.

[0187] In summary, the data presented above demonstrate: (1) a strategy has been developed that allows the rapid identification and isolation of natural antibodies without the need of generating phage display libraries; (2) a family of five clonally related broadly neutralizing antibodies has been described and their development tracked; (3) preliminary evidence of peripheral receptor editing in humans has been provided; (4) novel members of broadly neutralizing antibodies of the PG-like family have been described that are not genetically related to the previously described PG9 and PG16 broadly neutralizing antibodies; and (5) a method has been developed to increase accuracy of predicting putative reverted unmutated ancestors when more than a single monoclonal antibody is available.

[0188] For immunogen design for induction of quarternary V2, V3 antibodies, it is demonstrated in Example 5 that the gp120 Env A244 with a herpes simplex gD sequence can both bind well to the V2,V3 conformational determinant broad neutralizing Abs PG9, PG16, CH01-CH05, and also bind to reverted unmutated ancestors of CH01, 02 and 03 antibodies. Moreover, the 6240 transmitted founder Env can bind well to PG9, and PG16 mabs. Thus, a potent immunization regimen for induction of V2, V3 broad neutralizing antibodies is to prime several times (for example, from 1-3) with the A244 gp120 envelope with the gD sequence at the N-terminus and then boost, for example, with the 6240 transmitted founder gp140 (for example, from 1-3 times) either systemically (e.g., IM or subcutaneously) or mucosally (e.g., intranasally, sublingualy, intravaginally or rectally). Given the immunogenicity of the HSV receptor binding region in the A244 gp120, this construct containing the gD peptide can also be used for a HSV vaccine construct. Similarly, the gD peptide inserted at the N-terminus of any HIV-1 envelope in a similar manner can be used for inducing protective antibodies to herpes simplex virus types 1 and 2.

[0189] All documents and other information sources cited above are hereby incorporated in their entirety by reference.

Sequence CWU 1

1

96132PRTHuman immunodeficiency virus 1Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala 1 5 10 15 Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys 20 25 30 26PRTHomo sapiens 2Glu Leu Asp Lys Trp Ala 1 5 36PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Glu Leu Glu Lys Trp Ala 1 5 421DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 4tcgtcgtttt tcggtcgttt t 21543PRTHuman immunodeficiency virus 5Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly 1 5 10 15 Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp 20 25 30 Asn Ala Ser Trp Ser Asn Lys Ser Leu Asn Lys 35 40 620PRTHuman immunodeficiency virus 6Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala 1 5 10 15 Ser Leu Trp Asn 20 727PRTHerpes simplex virus 7Lys Tyr Ala Leu Val Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln 20 25 826PRTHomo sapiens 8Ala Arg Gly Thr Asp Tyr Thr Ile Asp Asp Ala Gly Ile His Tyr Gln 1 5 10 15 Gly Ser Gly Thr Phe Trp Tyr Phe Asp Leu 20 25 930PRTHuman immunodeficiency virus 9Glu Asn Lys Thr Ile Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu 1 5 10 15 Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 20 25 30 1030PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 10Glu Asn Lys Thr Ile Val Phe Asn His Ser Ser Gly Gly Ala Pro Ala 1 5 10 15 Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 20 25 30 116PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 11His His His His His His 1 5 12669PRTHuman immunodeficiency virus 12Met Arg Val Arg Gly Ile Trp Lys Asn Trp Pro Gln Trp Leu Ile Trp 1 5 10 15 Ser Ile Leu Gly Phe Trp Ile Gly Asn Met Glu Gly Ser Trp Val Thr 20 25 30 Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys Thr Thr Leu Phe 35 40 45 Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val His Asn Val Trp 50 55 60 Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Met Val 65 70 75 80 Leu Ala Asn Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asp Met Val 85 90 95 Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Glu Ser Leu Lys 100 105 110 Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Thr Asn 115 120 125 Val Lys Gly Asn Glu Ser Asp Thr Ser Glu Val Met Lys Asn Cys Ser 130 135 140 Phe Lys Ala Thr Thr Glu Leu Lys Asp Lys Lys His Lys Val His Ala 145 150 155 160 Leu Phe Tyr Lys Leu Asp Val Val Pro Leu Asn Gly Asn Ser Ser Ser 165 170 175 Ser Gly Glu Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile Thr Gln 180 185 190 Ala Cys Pro Lys Val Ser Phe Asp Pro Ile Pro Leu His Tyr Cys Ala 195 200 205 Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly 210 215 220 Thr Gly Pro Cys Arg Asn Val Ser Thr Val Gln Cys Thr His Gly Ile 225 230 235 240 Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu 245 250 255 Glu Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr 260 265 270 Ile Ile Val His Leu Asn Glu Ser Val Asn Ile Val Cys Thr Arg Pro 275 280 285 Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln Thr Phe 290 295 300 Tyr Ala Thr Gly Asp Ile Ile Gly Asn Ile Arg Gln Ala His Cys Asn 305 310 315 320 Ile Asn Glu Ser Lys Trp Asn Asn Thr Leu Gln Lys Val Gly Glu Glu 325 330 335 Leu Ala Lys His Phe Pro Ser Lys Thr Ile Lys Phe Glu Pro Ser Ser 340 345 350 Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys Arg Gly Glu 355 360 365 Phe Phe Tyr Cys Asn Thr Ser Asp Leu Phe Asn Gly Thr Tyr Arg Asn 370 375 380 Gly Thr Tyr Asn His Thr Gly Arg Ser Ser Asn Gly Thr Ile Thr Leu 385 390 395 400 Gln Cys Lys Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Arg 405 410 415 Ala Ile Tyr Ala Pro Pro Ile Glu Gly Glu Ile Thr Cys Asn Ser Asn 420 425 430 Ile Thr Gly Leu Leu Leu Leu Arg Asp Gly Gly Gln Ser Asn Glu Thr 435 440 445 Asn Asp Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 450 455 460 Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu 465 470 475 480 Gly Val Ala Pro Thr Glu Ala Lys Glu Arg Val Val Glu Arg Glu Lys 485 490 495 Glu Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 500 505 510 Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg 515 520 525 Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala 530 535 540 Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys 545 550 555 560 Gln Leu Gln Ala Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln 565 570 575 Gln Leu Leu Gly Met Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr 580 585 590 Ala Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Asn Glu Ile 595 600 605 Trp Gly Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile Asn Asn Tyr 610 615 620 Thr Asn Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu 625 630 635 640 Lys Asn Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp 645 650 655 Asn Trp Phe Asp Ile Ser Lys Trp Leu Trp Tyr Ile Lys 660 665 132022DNAHuman immunodeficiency virus 13atgcgcgtgc gcggcatctg gaagaactgg ccccagtggc tgatctggtc catcctgggc 60ttctggatcg gcaacatgga gggctcctgg gtgaccgtgt actacggcgt gcccgtgtgg 120aaggaggcca agaccaccct gttctgcgcc tccgacgcca aggcctacga gaaggaggtg 180cacaacgtgt gggccaccca cgcctgcgtg cccaccgacc ccaaccccca ggagatggtg 240ctggccaacg tgaccgagaa cttcaacatg tggaagaacg acatggtgga gcagatgcac 300gaggacatca tctccctgtg ggacgagtcc ctgaagccct gcgtgaagct gacccccctg 360tgcgtgaccc tgaactgcac caacgtgaag ggcaacgagt ccgacacctc cgaggtgatg 420aagaactgct ccttcaaggc caccaccgag ctgaaggaca agaagcacaa ggtgcacgcc 480ctgttctaca agctggacgt ggtgcccctg aacggcaact cctcctcctc cggcgagtac 540cgcctgatca actgcaacac ctccgccatc acccaggcct gccccaaggt gtccttcgac 600cccatccccc tgcactactg cgcccccgcc ggcttcgcca tcctgaagtg caacaacaag 660accttcaacg gcaccggccc ctgccgcaac gtgtccaccg tgcagtgcac ccacggcatc 720aagcccgtgg tgtccaccca gctgctgctg aacggctccc tggccgagga ggagatcatc 780atccgctccg agaacctgac caacaacgcc aagaccatca tcgtgcacct gaacgagtcc 840gtgaacatcg tgtgcacccg ccccaacaac aacacccgca agtccatccg catcggcccc 900ggccagacct tctacgccac cggcgacatc atcggcaaca tccgccaggc ccactgcaac 960atcaacgagt ccaagtggaa caacaccctg cagaaggtgg gcgaggagct ggccaagcac 1020ttcccctcca agaccatcaa gttcgagccc tcctccggcg gcgacctgga gatcaccacc 1080cactccttca actgccgcgg cgagttcttc tactgcaaca cctccgacct gttcaacggc 1140acctaccgca acggcaccta caaccacacc ggccgctcct ccaacggcac catcaccctg 1200cagtgcaaga tcaagcagat catcaacatg tggcaggagg tgggccgcgc catctacgcc 1260ccccccatcg agggcgagat cacctgcaac tccaacatca ccggcctgct gctgctgcgc 1320gacggcggcc agtccaacga gaccaacgac accgagacct tccgccccgg cggcggcgac 1380atgcgcgaca actggcgctc cgagctgtac aagtacaagg tggtggagat caagcccctg 1440ggcgtggccc ccaccgaggc caaggagcgc gtggtggagc gcgagaagga ggccgtgggc 1500atcggcgccg tgttcctggg cttcctgggc gccgccggct ccaccatggg cgccgcctcc 1560atgaccctga ccgtgcaggc ccgccagctg ctgtccggca tcgtgcagca gcagtccaac 1620ctgctgcgcg ccatcgaggc ccagcagcac atgctgcagc tgaccgtgtg gggcatcaag 1680cagctgcagg cccgcgtgct ggccatcgag cgctacctga aggaccagca gctgctgggc 1740atgtggggct gctccggcaa gctgatctgc accaccgccg tgccctggaa ctcctcctgg 1800tccaacaagt cccagaacga gatctggggc aacatgacct ggatgcagtg ggaccgcgag 1860atcaacaact acaccaacac catctaccgc ctgctggagg actcccagaa ccagcaggag 1920aagaacgaga aggacctgct ggccctggac tcctggaaga acctgtggaa ctggttcgac 1980atctccaagt ggctgtggta catcaagtag ggatcctcta ga 202214682PRTHuman immunodeficiency virus 14Met Arg Val Arg Gly Met Leu Arg Asn Cys Gln Gln Trp Trp Ile Trp 1 5 10 15 Gly Ile Leu Gly Phe Trp Met Leu Met Ile Cys Ser Val Val Gly Asn 20 25 30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys 35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr Glu Arg Glu Val 50 55 60 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80 Gln Glu Met Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95 Asn Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110 Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ile Leu 115 120 125 Glu Cys Asn Asn Ala Asn Gly Thr Thr Asn Asn Gly Ser Val Ile Val 130 135 140 Val Asn Glu Asn Ser Thr Met Tyr Gly Glu Ile Gln Asn Cys Ser Phe 145 150 155 160 Lys Val Asn Ser Glu Ile Lys Gly Lys Lys Gln Asp Val Tyr Ala Leu 165 170 175 Phe Asn Ser Leu Asp Ile Val Lys Leu Tyr Asn Asn Gly Thr Ser Gln 180 185 190 Tyr Arg Leu Ile Asn Cys Asn Thr Ser Thr Leu Thr Gln Ala Cys Pro 195 200 205 Lys Val Ser Phe Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly 210 215 220 Tyr Ala Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro 225 230 235 240 Cys Asn Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro Val 245 250 255 Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Gly Glu Ile 260 265 270 Ile Ile Arg Ser Lys Asn Leu Thr Asp Asn Thr Lys Thr Ile Ile Val 275 280 285 His Leu Asn Glu Ser Ile Lys Ile Asn Cys Ile Arg Pro Asn Asn Asn 290 295 300 Thr Arg Arg Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala Ala 305 310 315 320 Asn Gly Ile Val Gly Asn Ile Arg Gln Ala His Cys Asn Ile Ser Glu 325 330 335 Gly Glu Trp Asn Lys Thr Leu Tyr Arg Val Ser Arg Lys Leu Ala Glu 340 345 350 His Phe Pro Gly Lys Glu Ile Lys Phe Lys Pro His Ser Gly Gly Asp 355 360 365 Leu Glu Ile Thr Thr His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr 370 375 380 Cys Asn Thr Ser Lys Leu Phe Asn Gly Thr Tyr Asn Gly Thr Tyr Thr 385 390 395 400 Asn Asn Asp Thr Asn Ser Thr Ile Ile Leu Pro Cys Arg Ile Lys Gln 405 410 415 Ile Ile Asn Met Trp Gln Glu Val Gly Gln Ala Met Tyr Ala Pro Pro 420 425 430 Ile Glu Gly Ile Ile Ala Cys Asn Ser Thr Ile Thr Gly Leu Leu Leu 435 440 445 Thr Arg Asp Gly Gly Asp Lys Asn Gly Ser Lys Pro Glu Ile Phe Arg 450 455 460 Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys 465 470 475 480 Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Ile Ala Pro Thr Lys Ala 485 490 495 Lys Glu Arg Val Val Glu Lys Glu Lys Thr Ile Gln Lys Glu Ala Val 500 505 510 Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr 515 520 525 Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu 530 535 540 Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala 545 550 555 560 Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln 565 570 575 Ala Arg Val Leu Ala Met Glu Arg Tyr Leu Gln Asp Gln Gln Leu Leu 580 585 590 Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro 595 600 605 Trp Asn Ser Ser Trp Ser Asn Lys Thr Leu Glu Tyr Ile Trp Gly Asn 610 615 620 Met Thr Trp Met Gln Trp Asp Arg Glu Ile Asp Asn Tyr Thr Gly Ile 625 630 635 640 Ile Tyr Asp Leu Leu Glu Asp Ser Gln Ile Gln Gln Glu Lys Asn Glu 645 650 655 Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp Ser Trp Phe 660 665 670 Ser Ile Thr Asn Trp Leu Trp Tyr Ile Lys 675 680 152046DNAHuman immunodeficiency virus 15atgcgcgtgc gcggcatgct gcgcaactgc cagcagtggt ggatctgggg catcctgggc 60ttctggatgc tgatgatctg ctccgtggtg ggcaacctgt gggtgaccgt gtactacggc 120gtgcccgtgt ggaaggaggc caagaccacc ctgttctgcg cctccgacgc ccgcgcctac 180gagcgcgagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240caggagatgg tgctggtgaa cgtgaccgag aacttcaaca tgtggaagaa cgacatggtg 300gaccagatgc acgaggacat catctccctg tgggaccagt ccctgaagcc ctgcgtgaag 360ctgacccccc tgtgcgtgat cctggagtgc aacaacgcca acggcaccac caacaacggc 420tccgtgatcg tggtgaacga gaactccacc atgtacggcg agatccagaa ctgctccttc 480aaggtgaact ccgagatcaa gggcaagaag caggacgtgt acgccctgtt caactccctg 540gacatcgtga agctgtacaa caacggcacc tcccagtacc gcctgatcaa ctgcaacacc 600tccaccctga cccaggcctg ccccaaggtg tccttcgacc ccatccccat ccactactgc 660gcccccgccg gctacgccat cctgaagtgc aacaacaaga ccttcaacgg caccggcccc 720tgcaacaacg tgtccaccgt gcagtgcacc cacggcatca agcccgtggt gtccacccag 780ctgctgctga acggctccct ggccgagggc gagatcatca tccgctccaa gaacctgacc 840gacaacacca agaccatcat cgtgcacctg aacgagtcca tcaagatcaa ctgcatccgc 900cccaacaaca acacccgccg ctccatccgc atcggccccg gccaggcctt ctacgccgcc 960aacggcatcg tgggcaacat ccgccaggcc cactgcaaca tctccgaggg cgagtggaac 1020aagaccctgt accgcgtgtc ccgcaagctg gccgagcact tccccggcaa ggagatcaag 1080ttcaagcccc actccggcgg cgacctggag atcaccaccc actccttcaa ctgccgcggc 1140gagttcttct actgcaacac ctccaagctg ttcaacggca cctacaacgg cacctacacc 1200aacaacgaca ccaactccac catcatcctg ccctgccgca tcaagcagat catcaacatg 1260tggcaggagg tgggccaggc catgtacgcc ccccccatcg agggcatcat cgcctgcaac 1320tccaccatca ccggcctgct gctgacccgc gacggcggcg acaagaacgg ctccaagccc 1380gagatcttcc gccccggcgg cggcgacatg cgcgacaact ggcgctccga gctgtacaag 1440tacaaggtgg tggagatcaa gcccctgggc atcgccccca ccaaggccaa ggagcgcgtg 1500gtggagaagg agaagaccat ccagaaggag gccgtgggca tcggcgccgt gttcctgggc 1560ttcctgggcg ccgccggctc caccatgggc gccgcctcca tcaccctgac cgtgcaggcc 1620cgccagctgc tgtccggcat cgtgcagcag cagtccaacc tgctgcgcgc catcgaggcc 1680cagcagcaca tgctgcagct gaccgtgtgg ggcatcaagc agctgcaggc ccgcgtgctg 1740gccatggagc gctacctgca ggaccagcag ctgctgggca tctggggctg ctccggcaag 1800ctgatctgca ccaccgccgt gccctggaac tcctcctggt ccaacaagac cctggagtac 1860atctggggca acatgacctg gatgcagtgg gaccgcgaga tcgacaacta caccggcatc 1920atctacgacc tgctggagga ctcccagatc cagcaggaga agaacgagaa ggacctgctg 1980gccctggact cctggaagaa cctgtggtcc tggttctcca tcaccaactg gctgtggtac 2040atcaag

204616673PRTHuman immunodeficiency virus 16Met Arg Val Met Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Glu 1 5 10 15 Gly Ile Leu Leu Leu Gly Ile Leu Met Ile Cys Ser Ala Ala Asp Asn 20 25 30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Arg Glu Ala Thr 35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Ala 50 55 60 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80 Gln Glu Val Glu Leu Lys Asn Val Thr Glu Asn Phe Asn Met Trp Glu 85 90 95 Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110 Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125 Asn Cys Thr Asp Leu Gly Asn Val Thr Asn Thr Thr Asn Ser Asn Gly 130 135 140 Glu Met Met Glu Lys Gly Glu Val Lys Asn Cys Ser Phe Lys Ile Thr 145 150 155 160 Thr Asp Ile Lys Asp Arg Thr Arg Lys Glu Tyr Ala Leu Phe Tyr Lys 165 170 175 Leu Asp Val Val Pro Ile Asn Asp Thr Arg Tyr Arg Leu Val Ser Cys 180 185 190 Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro 195 200 205 Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys 210 215 220 Asn Asp Lys Gln Phe Ile Gly Thr Gly Pro Cys Thr Asn Val Ser Thr 225 230 235 240 Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu 245 250 255 Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile Arg Ser Val Asn 260 265 270 Phe Ser Asp Asn Ala Lys Thr Ile Ile Val Gln Leu Asn Lys Ser Val 275 280 285 Glu Ile Thr Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Pro 290 295 300 Met Gly Pro Gly Lys Ala Phe Tyr Ala Arg Gly Asp Ile Thr Gly Asp 305 310 315 320 Ile Arg Lys Ala Tyr Cys Glu Ile Asn Gly Thr Glu Trp His Ser Thr 325 330 335 Leu Lys Leu Val Val Glu Lys Leu Arg Glu Gln Tyr Asn Lys Thr Ile 340 345 350 Val Phe Asn Arg Ser Ser Gly Gly Asp Pro Glu Ile Val Met Tyr Ser 355 360 365 Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Lys Leu Phe 370 375 380 Asn Ser Thr Trp Pro Trp Asn Asp Thr Lys Gly Ser His Asp Thr Asn 385 390 395 400 Gly Thr Leu Ile Leu Pro Cys Lys Ile Lys Gln Ile Ile Asn Met Trp 405 410 415 Gln Gly Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Glu Gly Lys Ile 420 425 430 Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 435 440 445 Tyr Glu Ser Asn Glu Thr Asp Glu Ile Phe Arg Pro Gly Gly Gly Asp 450 455 460 Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys 465 470 475 480 Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Glu Arg Val Val 485 490 495 Gln Arg Glu Lys Glu Ala Phe Gly Leu Gly Ala Val Phe Leu Gly Phe 500 505 510 Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr 515 520 525 Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn 530 535 540 Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val 545 550 555 560 Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr 565 570 575 Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu 580 585 590 Ile Cys Thr Thr Thr Val Pro Trp Asn Thr Ser Trp Ser Asn Lys Ser 595 600 605 Leu Glu Gln Ile Trp Asp Asn Met Thr Trp Met Glu Trp Glu Arg Glu 610 615 620 Ile Asp Asn Tyr Thr Gly Tyr Ile Tyr Gln Leu Ile Glu Glu Ser Gln 625 630 635 640 Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Ala Leu Asp Lys Trp 645 650 655 Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile 660 665 670 Lys 172019DNAHuman immunodeficiency virus 17atgcgcgtga tgggcatccg caagaactac cagcacctgt ggcgcgaggg catcctgctg 60ctgggcatcc tgatgatctg ctccgccgcc gacaacctgt gggtgaccgt gtactacggc 120gtgcccgtgt ggcgcgaggc caccaccacc ctgttctgcg cctccgacgc caaggcctac 180gacaccgagg cccacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240caggaggtgg agctgaagaa cgtgaccgag aacttcaaca tgtgggagaa caacatggtg 300gagcagatgc acgaggacat catctccctg tgggaccagt ccctgaagcc ctgcgtgaag 360ctgacccccc tgtgcgtgac cctgaactgc accgacctgg gcaacgtgac caacaccacc 420aactccaacg gcgagatgat ggagaagggc gaggtgaaga actgctcctt caagatcacc 480accgacatca aggaccgcac ccgcaaggag tacgccctgt tctacaagct ggacgtggtg 540cccatcaacg acacccgcta ccgcctggtg tcctgcaaca cctccgtgat cacccaggcc 600tgccccaagg tgtccttcga gcccatcccc atccactact gcgcccccgc cggcttcgcc 660atcctgaagt gcaacgacaa gcagttcatc ggcaccggcc cctgcaccaa cgtgtccacc 720gtgcagtgca cccacggcat ccgccccgtg gtgtccaccc agctgctgct gaacggctcc 780ctggccgagg aggaggtggt gatccgctcc gtgaacttct ccgacaacgc caagaccatc 840atcgtgcagc tgaacaagtc cgtggagatc acctgcaccc gccccaacaa caacacccgc 900aagtccatcc ccatgggccc cggcaaggcc ttctacgccc gcggcgacat caccggcgac 960atccgcaagg cctactgcga gatcaacggc accgagtggc actccaccct gaagctggtg 1020gtggagaagc tgcgcgagca gtacaacaag accatcgtgt tcaaccgctc ctccggcggc 1080gaccccgaga tcgtgatgta ctccttcaac tgcggcggcg agttcttcta ctgcaactcc 1140accaagctgt tcaactccac ctggccctgg aacgacacca agggctccca cgacaccaac 1200ggcaccctga tcctgccctg caagatcaag cagatcatca acatgtggca gggcgtgggc 1260aaggccatgt acgccccccc catcgagggc aagatccgct gctcctccaa catcaccggc 1320ctgctgctga cccgcgacgg cggctacgag tccaacgaga ccgacgagat cttccgcccc 1380ggcggcggcg acatgcgcga caactggcgc tccgagctgt acaagtacaa ggtggtgaag 1440atcgagcccc tgggcgtggc ccccaccaag gccaaggagc gcgtggtgca gcgcgagaag 1500gaggccttcg gcctgggcgc cgtgttcctg ggcttcctgg gcgccgccgg ctccaccatg 1560ggcgccgcct ccatcaccct gaccgtgcag gcccgccagc tgctgtccgg catcgtgcag 1620cagcagaaca acctgctgcg cgccatcgag gcccagcagc acctgctgca gctgaccgtg 1680tggggcatca agcagctgca ggcccgcgtg ctggccgtgg agcgctacct gaaggaccag 1740cagctgctgg gcatctgggg ctgctccggc aagctgatct gcaccaccac cgtgccctgg 1800aacacctcct ggtccaacaa gtccctggag cagatctggg acaacatgac ctggatggag 1860tgggagcgcg agatcgacaa ctacaccggc tacatctacc agctgatcga ggagtcccag 1920aaccagcagg agaagaacga gcaggagctg ctggccctgg acaagtgggc ctccctgtgg 1980aactggttcg acatcaccaa ctggctgtgg tacatcaag 201918693PRTHuman immunodeficiency virus 18Met Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trp 1 5 10 15 Gly Thr Met Leu Leu Gly Ile Leu Met Ile Cys Ser Ala Ala Ala Gln 20 25 30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val 50 55 60 His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80 Gln Glu Leu Val Leu Ala Asn Val Thr Glu Asn Phe Asn Met Trp Asn 85 90 95 Asn Thr Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110 Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125 Asn Cys Thr Asp Val Thr Asn Ala Thr Asn Ile Asn Ala Thr Asn Ile 130 135 140 Asn Asn Ser Ser Gly Gly Val Glu Ser Gly Glu Ile Lys Asn Cys Ser 145 150 155 160 Phe Asn Ile Thr Thr Ser Val Arg Asp Lys Val Gln Lys Glu Tyr Ala 165 170 175 Leu Phe Tyr Lys Leu Asp Ile Val Pro Ile Thr Asn Glu Ser Ser Lys 180 185 190 Tyr Arg Leu Ile Ser Cys Asn Thr Ser Val Leu Thr Gln Ala Cys Pro 195 200 205 Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly 210 215 220 Phe Ala Ile Leu Lys Cys Asn Asn Glu Thr Phe Asn Gly Lys Gly Pro 225 230 235 240 Cys Ile Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val 245 250 255 Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Lys Glu Val 260 265 270 Ile Ile Arg Ser Asp Asn Phe Ser Asp Asn Ala Lys Asn Ile Ile Val 275 280 285 Gln Leu Lys Glu Tyr Val Lys Ile Asn Cys Thr Arg Pro Asn Asn Asn 290 295 300 Thr Arg Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr 305 310 315 320 Gly Glu Ile Ile Gly Asn Ile Arg Gln Ala His Cys Asn Ile Ser Arg 325 330 335 Ser Lys Trp Asn Asp Thr Leu Lys Gln Ile Ala Ala Lys Leu Gly Glu 340 345 350 Gln Phe Arg Asn Lys Thr Ile Val Phe Asn Pro Ser Ser Gly Gly Asp 355 360 365 Leu Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 370 375 380 Cys Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp Ile Arg Glu Gly Asn 385 390 395 400 Asn Gly Thr Trp Asn Gly Thr Ile Gly Leu Asn Asp Thr Ala Gly Asn 405 410 415 Asp Thr Ile Ile Leu Pro Cys Lys Ile Lys Gln Ile Ile Asn Met Trp 420 425 430 Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile 435 440 445 Arg Cys Ser Ser Asn Ile Thr Gly Leu Ile Leu Thr Arg Asp Gly Gly 450 455 460 Lys Asp Asp Ser Asn Gly Ser Glu Ile Leu Glu Ile Phe Arg Pro Gly 465 470 475 480 Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 485 490 495 Val Val Arg Ile Glu Pro Leu Gly Val Ala Pro Thr Arg Ala Arg Glu 500 505 510 Arg Val Val Gln Lys Glu Lys Glu Ala Val Gly Leu Gly Ala Met Phe 515 520 525 Leu Gly Phe Leu Gly Ala Ala Gly Ser Ala Met Gly Ala Ala Ser Met 530 535 540 Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln 545 550 555 560 Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Met Leu Gln 565 570 575 Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val 580 585 590 Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser 595 600 605 Gly Lys Leu Ile Cys Thr Thr Asp Val Pro Trp Asp Thr Ser Trp Ser 610 615 620 Asn Lys Thr Leu Asp Asp Ile Trp Gly Ser Asn Met Thr Trp Met Glu 625 630 635 640 Trp Glu Arg Glu Ile Asp Asn Tyr Thr Ser Thr Ile Tyr Thr Leu Leu 645 650 655 Glu Glu Ala Gln Tyr Gln Gln Glu Lys Asn Glu Lys Glu Leu Leu Glu 660 665 670 Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp 675 680 685 Leu Trp Tyr Ile Arg 690 192088DNAHuman immunodeficiency virus 19atgcgcgtga agggcatccg caagaactac cagcacctgt ggcgctgggg caccatgctg 60ctgggcatcc tgatgatctg ctccgccgcc gcccagctgt gggtgaccgt gtactacggc 120gtgcccgtgt ggaaggaggc caccaccacc ctgttctgcg cctccgacgc caaggcctac 180gacaccgagg tgcacaacgt gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240caggagctgg tgctggccaa cgtgaccgag aacttcaaca tgtggaacaa caccatggtg 300gagcagatgc acgaggacat catctccctg tgggaccagt ccctgaagcc ctgcgtgaag 360ctgacccccc tgtgcgtgac cctgaactgc accgacgtga ccaacgccac caacatcaac 420gccaccaaca tcaacaactc ctccggcggc gtggagtccg gcgagatcaa gaactgctcc 480ttcaacatca ccacctccgt gcgcgacaag gtgcagaagg agtacgccct gttctacaag 540ctggacatcg tgcccatcac caacgagtcc tccaagtacc gcctgatctc ctgcaacacc 600tccgtgctga cccaggcctg ccccaaggtg tccttcgagc ccatccccat ccactactgc 660gcccccgccg gcttcgccat cctgaagtgc aacaacgaga ccttcaacgg caagggcccc 720tgcatcaacg tgtccaccgt gcagtgcacc cacggcatcc gccccgtggt gtccacccag 780ctgctgctga acggctccct ggccgagaag gaggtgatca tccgctccga caacttctcc 840gacaacgcca agaacatcat cgtgcagctg aaggagtacg tgaagatcaa ctgcacccgc 900cccaacaaca acacccgcaa gtccatccac atcggccccg gccgcgcctt ctacgccacc 960ggcgagatca tcggcaacat ccgccaggcc cactgcaaca tctcccgctc caagtggaac 1020gacaccctga agcagatcgc cgccaagctg ggcgagcagt tccgcaacaa gaccatcgtg 1080ttcaacccct cctccggcgg cgacctggag atcgtgaccc actccttcaa ctgcggcggc 1140gagttcttct actgcaacac caccaagctg ttcaactcca cctggatccg cgagggcaac 1200aacggcacct ggaacggcac catcggcctg aacgacaccg ccggcaacga caccatcatc 1260ctgccctgca agatcaagca gatcatcaac atgtggcagg aggtgggcaa ggccatgtac 1320gcccccccca tccgcggcca gatccgctgc tcctccaaca tcaccggcct gatcctgacc 1380cgcgacggcg gcaaggacga ctccaacggc tccgagatcc tggagatctt ccgccccggc 1440ggcggcgaca tgcgcgacaa ctggcgctcc gagctgtaca agtacaaggt ggtgcgcatc 1500gagcccctgg gcgtggcccc cacccgcgcc cgcgagcgcg tggtgcagaa ggagaaggag 1560gccgtgggcc tgggcgccat gttcctgggc ttcctgggcg ccgccggctc cgccatgggc 1620gccgcctcca tgaccctgac cgtgcaggcc cgccagctgc tgtccggcat cgtgcagcag 1680cagaacaacc tgctgcgcgc catcgaggcc cagcagcaca tgctgcagct gaccgtgtgg 1740ggcatcaagc agctgcaggc ccgcgtgctg gccgtggagc gctacctgaa ggaccagcag 1800ctgctgggca tctggggctg ctccggcaag ctgatctgca ccaccgacgt gccctgggac 1860acctcctggt ccaacaagac cctggacgac atctggggct ccaacatgac ctggatggag 1920tgggagcgcg agatcgacaa ctacacctcc accatctaca ccctgctgga ggaggcccag 1980taccagcagg agaagaacga gaaggagctg ctggagctgg acaagtgggc ctccctgtgg 2040aactggttcg acatcaccaa ctggctgtgg tacatccgct agggatcc 2088201902DNAHuman immunodeficiency virus 20atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60tccgtgctag ctgtggagaa gctgtgggtg actgtatact atggggtgcc tgtgtggaag 120gaggccacca ccaccctgtt ctgtgcctct gatgccaagg cctatgacac tgaggtccac 180aatgtctggg ccacccatgc ctgtgtgccc actgacccca accctcagga ggtggtgctg 240gagaatgtga ctgagcactt caacatgtgg aagaacaaca tggtggagca gatgcaggag 300gacatcatca gcctgtggga ccagagcctg aagccctgtg tgaagctgac ccccctgtgt 360gtgaccctga actgcaagga tgtgaacgcc accaacacca ccaatgactc tgagggcact 420atggagaggg gtgagatcaa gaactgcagc ttcaacatca ccaccagcat cagggatgag 480gtgcagaagg agtatgccct gttctacaag ctggatgtgg tgcccattga caacaacaac 540accagctaca ggctgatcag ctgtgacacc tctgtgatca cccaggcctg ccccaagatc 600agctttgagc ccatccccat ccactactgt gcccctgctg gctttgccat cctgaagtgc 660aatgacaaga ccttcaatgg caaaggccct tgcaagaatg tgagcactgt gcagtgcact 720catggcatca ggcctgtggt gagcacccag ctgctgctga atggcagcct ggctgaggag 780gaggtggtga tcaggtctga caacttcacc aacaatgcca agaccatcat tgtgcagctg 840aaggagtctg tggagatcaa ctgcaccagg cccaacaaca acaccaggaa gagcattcac 900attggccctg gcagggcctt ctacaccact ggggagatca ttggggacat caggcaggcc 960cactgcaaca tcagcagggc caagtggaat gacaccctga agcagattgt gatcaagctg 1020agggagcagt ttgagaacaa gaccattgtg ttcaatcaca gctctggtgg tgatcctgag 1080attgtgatgc acagcttcaa ctgtggtggt gagttcttct actgcaacag cacccagctg 1140ttcaacagca cctggaacaa caacactgag ggcagcaaca acactgaggg caacaccatc 1200accctgcctt gcaggatcaa gcagatcatc aacatgtggc aggaggtggg caaggccatg 1260tatgctcctc ccatcagggg ccagatcagg tgcagcagca acatcactgg cctgctgctg 1320accagggatg gtggcatcaa tgagaatggc actgagattt tcaggcctgg tggtggggac 1380atgagggaca actggaggtc tgagctgtac aagtacaagg tggtgaagat tgagcccctt 1440ggtgtggctc ccaccaaggc taagaccctg actgtgcagg ccaggctgct gctgtctggc 1500attgtgcagc agcagaacaa cctgctgagg gccattgagg ctcaacagag gatgctccag 1560ctcactgtct ggggcatcaa gcagctccag gccagggtgc tggctgtgga gaggtatctt 1620ggggatcagc agctccttgg catctggggc tgctctggca agctgatctg caccactgct 1680gtgccctgga atgccagctg gagcaacaag agcctggaca ggatctggaa caacatgacc 1740tggatggagt gggagaggga gattgacaac tacacctctg

agatttacac cctgattgag 1800gagagccaga accagcagga gaagaatgag caggagctgc tggagctgga caagtgggcc 1860agcctgtgga actggtttga catcaccaag tggctgtggt ag 190221633PRTHuman immunodeficiency virus 21Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Val Leu Ala Val Glu Lys Leu Trp Val Thr Val 20 25 30 Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys 35 40 45 Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala 50 55 60 Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Val Val Leu 65 70 75 80 Glu Asn Val Thr Glu His Phe Asn Met Trp Lys Asn Asn Met Val Glu 85 90 95 Gln Met Gln Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro 100 105 110 Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Lys Asp Val 115 120 125 Asn Ala Thr Asn Thr Thr Asn Asp Ser Glu Gly Thr Met Glu Arg Gly 130 135 140 Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Ser Ile Arg Asp Glu 145 150 155 160 Val Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val Pro Ile 165 170 175 Asp Asn Asn Asn Thr Ser Tyr Arg Leu Ile Ser Cys Asp Thr Ser Val 180 185 190 Ile Thr Gln Ala Cys Pro Lys Ile Ser Phe Glu Pro Ile Pro Ile His 195 200 205 Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asp Lys Thr 210 215 220 Phe Asn Gly Lys Gly Pro Cys Lys Asn Val Ser Thr Val Gln Cys Thr 225 230 235 240 His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser 245 250 255 Leu Ala Glu Glu Glu Val Val Ile Arg Ser Asp Asn Phe Thr Asn Asn 260 265 270 Ala Lys Thr Ile Ile Val Gln Leu Lys Glu Ser Val Glu Ile Asn Cys 275 280 285 Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile His Ile Gly Pro Gly 290 295 300 Arg Ala Phe Tyr Thr Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln Ala 305 310 315 320 His Cys Asn Ile Ser Arg Ala Lys Trp Asn Asp Thr Leu Lys Gln Ile 325 330 335 Val Ile Lys Leu Arg Glu Gln Phe Glu Asn Lys Thr Ile Val Phe Asn 340 345 350 His Ser Ser Gly Gly Asp Pro Glu Ile Val Met His Ser Phe Asn Cys 355 360 365 Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr 370 375 380 Trp Asn Asn Asn Thr Glu Gly Ser Asn Asn Thr Glu Gly Asn Thr Ile 385 390 395 400 Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val 405 410 415 Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln Ile Arg Cys Ser 420 425 430 Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Ile Asn Glu 435 440 445 Asn Gly Thr Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn 450 455 460 Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu 465 470 475 480 Gly Val Ala Pro Thr Lys Ala Lys Thr Leu Thr Val Gln Ala Arg Leu 485 490 495 Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 500 505 510 Glu Ala Gln Gln Arg Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln 515 520 525 Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gly Asp Gln Gln 530 535 540 Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 545 550 555 560 Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Arg Ile Trp 565 570 575 Asn Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr 580 585 590 Ser Glu Ile Tyr Thr Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 595 600 605 Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn 610 615 620 Trp Phe Asp Ile Thr Lys Trp Leu Trp 625 630 222082DNAHuman immunodeficiency virus 22atgcgcgtga agggcatccg caagaactac cagcacctgt ggcgctgggg catctggcgc 60tggggcatca tgctgctggg caccctgatg atctgctccg ccaccgagaa gctgtgggtg 120accgtgtact acggcgtgcc cgtgtggaag gaggccacca ccaccctgtt ctgcgcctcc 180gacgccaagg cctactcccc cgagaagcac aacatctggg ccacccacgc ctgcgtgccc 240accgacccca acccccagga gctggtgctg ggcaacgtga ccgaggactt caacatgtgg 300aagaacaaca tggtggagca gatgcacgag gacatcatct ccctgtggga ccagtccctg 360aagccctgcg tgaagctgac ccccctgtgc gtgaccctga actgcaccga cctgaagaac 420tccgccaccg acaccaacgg cacctccggc accaacaacc gcaccgtgga gcagggcatg 480gagaccgaga tcaagaactg ctccttcaac atcaccaccg gcatcggcaa caagatgcag 540aaggagtacg ccctgttcta caagctggac gtggtgccca tcgactccaa caacaactcc 600gacaacacct cctaccgcct gatctcctgc aacacctccg tggtgaccca ggcctgcccc 660aagacctcct tcgagcccat ccccatccac tactgcgccc ccgccggctt cgccatcctg 720aagtgcaaca acaagacctt ctccggcaag ggcccctgca agaacgtgtc caccgtgcag 780tgcacccacg gcatccgccc cgtggtgtcc acccagctgc tgctgaacgg ctccctggcc 840gaggaggaga tcgtgatccg ctccgagaac ttcaccaaca acgccaagac catcatcgtg 900cagctgaacg agtccgtgat catcaactgc acccgcccca acaacaacac ccgcaagggc 960atccacatcg gcctgggccg cgccctgtac gccaccggcg acatcatcgg cgacatccgc 1020caggcccact gcaacctgtc ctccaagtcc tggaacaaga ccctgcagca ggtggtgcgc 1080aagctgcgcg agcagttcgg caacaagacc atcgccttca accagtcctc cggcggcgac 1140caggagatcg tgaagcactc cttcaactgc ggcggcgagt tcttctactg cgacaccacc 1200cagctgttca actccacctg gtcctccaac gacacctgga actccaccgg cgtgcaggac 1260aacaacatca ccctgccctg ccgcatcaag cagatcatca acatgtggca ggaggtgggc 1320aaggccatgt acgccccccc catccagggc ctgatctcct gctcctccaa catcaccggc 1380ctgctgctga cccgcgacgg cggcaccaac aacaccaacg ccaccgagat cttccgcccc 1440ggcggcggcg acatgcgcga caactggcgc tccgagctgt acaagtacaa ggtggtgaag 1500atcgagcccc tgggcatcgc ccccaccaag gccaaggagc gcgtggtgca gcgcgagaag 1560gaggccgtgg gcctgggcgc cgtgttcatc ggcttcctgg gcgccgccgg ctccaccatg 1620ggcgccgcct ccgtgaccct gaccgtgcag gcccgccagc tgctgtccgg catcgtgcag 1680cagcagaaca acctgctgcg cgccatcgag gcccagcagc acatgctgca gctgaccgtg 1740tggggcatca agcagctgca ggcccgcatc ctggccgtgg agcgctacct gaaggaccag 1800cagatcctgg gcatctgggg ctgctccggc aagctgatct gccccaccgc cgtgccctgg 1860aacgcctcct ggtccaacaa gtccctgacc gccatctgga acaacatgac ctggatggag 1920tgggagcgcg agatcgacaa ctacaccggc ctgatctact ccctgatcga ggagtcccag 1980atccagcagg agcagaacga gaaggagctg ctggagctgg acaagtgggc ctccctgtgg 2040aactggttcg acatcaccaa gtggctgtgg tacatcaagt ag 208223693PRTHuman immunodeficiency virus 23Met Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trp 1 5 10 15 Gly Ile Trp Arg Trp Gly Ile Met Leu Leu Gly Thr Leu Met Ile Cys 20 25 30 Ser Ala Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val 35 40 45 Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala 50 55 60 Tyr Ser Pro Glu Lys His Asn Ile Trp Ala Thr His Ala Cys Val Pro 65 70 75 80 Thr Asp Pro Asn Pro Gln Glu Leu Val Leu Gly Asn Val Thr Glu Asp 85 90 95 Phe Asn Met Trp Lys Asn Asn Met Val Glu Gln Met His Glu Asp Ile 100 105 110 Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro 115 120 125 Leu Cys Val Thr Leu Asn Cys Thr Asp Leu Lys Asn Ser Ala Thr Asp 130 135 140 Thr Asn Gly Thr Ser Gly Thr Asn Asn Arg Thr Val Glu Gln Gly Met 145 150 155 160 Glu Thr Glu Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Gly Ile Gly 165 170 175 Asn Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys Leu Asp Val Val 180 185 190 Pro Ile Asp Ser Asn Asn Asn Ser Asp Asn Thr Ser Tyr Arg Leu Ile 195 200 205 Ser Cys Asn Thr Ser Val Val Thr Gln Ala Cys Pro Lys Thr Ser Phe 210 215 220 Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu 225 230 235 240 Lys Cys Asn Asn Lys Thr Phe Ser Gly Lys Gly Pro Cys Lys Asn Val 245 250 255 Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln 260 265 270 Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile Val Ile Arg Ser 275 280 285 Glu Asn Phe Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Asn Glu 290 295 300 Ser Val Ile Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly 305 310 315 320 Ile His Ile Gly Leu Gly Arg Ala Leu Tyr Ala Thr Gly Asp Ile Ile 325 330 335 Gly Asp Ile Arg Gln Ala His Cys Asn Leu Ser Ser Lys Ser Trp Asn 340 345 350 Lys Thr Leu Gln Gln Val Val Arg Lys Leu Arg Glu Gln Phe Gly Asn 355 360 365 Lys Thr Ile Ala Phe Asn Gln Ser Ser Gly Gly Asp Gln Glu Ile Val 370 375 380 Lys His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asp Thr Thr 385 390 395 400 Gln Leu Phe Asn Ser Thr Trp Ser Ser Asn Asp Thr Trp Asn Ser Thr 405 410 415 Gly Val Gln Asp Asn Asn Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile 420 425 430 Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile 435 440 445 Gln Gly Leu Ile Ser Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr 450 455 460 Arg Asp Gly Gly Thr Asn Asn Thr Asn Ala Thr Glu Ile Phe Arg Pro 465 470 475 480 Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 485 490 495 Lys Val Val Lys Ile Glu Pro Leu Gly Ile Ala Pro Thr Lys Ala Lys 500 505 510 Glu Arg Val Val Gln Arg Glu Lys Glu Ala Val Gly Leu Gly Ala Val 515 520 525 Phe Ile Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser 530 535 540 Val Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln 545 550 555 560 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Met Leu 565 570 575 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala 580 585 590 Val Glu Arg Tyr Leu Lys Asp Gln Gln Ile Leu Gly Ile Trp Gly Cys 595 600 605 Ser Gly Lys Leu Ile Cys Pro Thr Ala Val Pro Trp Asn Ala Ser Trp 610 615 620 Ser Asn Lys Ser Leu Thr Ala Ile Trp Asn Asn Met Thr Trp Met Glu 625 630 635 640 Trp Glu Arg Glu Ile Asp Asn Tyr Thr Gly Leu Ile Tyr Ser Leu Ile 645 650 655 Glu Glu Ser Gln Ile Gln Gln Glu Gln Asn Glu Lys Glu Leu Leu Glu 660 665 670 Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Lys Trp 675 680 685 Leu Trp Tyr Ile Lys 690 2415PRTHomo sapiens 24Tyr Leu Glu Glu Glu Leu Asp Lys Trp Ala Lys Ile Ala Ala Tyr 1 5 10 15 2515PRTMus musculus 25Tyr Leu Glu Glu Glu Leu Asp Lys Trp Ala Lys Met Gly Ala Tyr 1 5 10 15 2625PRTHomo sapiens 26Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Ile Ala Ala 20 25 2725PRTPan troglodytes 27Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Ile Ala Ala 20 25 2825PRTMus musculus 28Ser Leu Gly Leu Gln Pro Lys Met Val Arg Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Ala 20 25 2925PRTAiluropoda melanoleuca 29Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Ala 20 25 3025PRTBos taurus 30Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Ala 20 25 3125PRTOryctolagus cuniculus 31Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Ala 20 25 3225PRTEquus caballus 32Ser Leu Gly Leu Gln Pro Lys Met Val Lys Thr Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Gly 20 25 3325PRTMonodelphis domestica 33Ser Leu Gly Leu Gln Pro Arg Asn Val Lys Lys Tyr Leu Glu Glu Glu 1 5 10 15 Leu Glu Lys Trp Ala Lys Met Gly Gly 20 25 3425PRTOrnithorhynchus anatinus 34Ser Leu Gly Leu Gln Pro Lys Lys Val Lys Ala Tyr Leu Glu Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Lys Met Gly Ala 20 25 3525PRTGallus gallus 35Ser Leu Gly Leu Gln Pro Lys Lys Val Lys Thr Tyr Leu Asp Glu Glu 1 5 10 15 Leu Asp Lys Trp Ala Arg Thr Gly Val 20 25 3625PRTDanio rerio 36Ser Leu Gly Leu Gln Pro Lys Asn Thr Lys Lys Tyr Ile Asp Glu Glu 1 5 10 15 Leu Glu Lys Trp Ala Lys Thr Gly Val 20 25 3736PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 37Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Leu 1 5 10 15 Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser 20 25 30 Leu Trp Asn Phe 35 3827PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp 1 5 10 15 Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys 20 25 391332PRTEscherichia coli 39Met Val Tyr Ser Tyr Thr Glu Lys Lys Arg Ile Arg Lys Asp Phe Gly 1 5 10 15 Lys Arg Pro Gln Val Leu Asp Val Pro Tyr Leu Leu Ser Ile Gln Leu 20 25 30 Asp Ser Phe Gln Lys Phe Ile Glu Gln Asp Pro Glu Gly Gln Tyr Gly 35 40 45 Leu Glu Ala Ala Phe Arg Ser Val Phe Pro Ile Gln Ser Tyr Ser Gly 50 55 60 Asn Ser Glu Leu Gln Tyr Val Ser Tyr Arg Leu Gly Glu Pro Val Phe 65 70 75 80 Asp Val Gln Glu Cys Gln Ile Arg Gly Val Thr Tyr Ser Ala Pro Leu 85 90 95 Arg Val Lys Leu Arg Leu Val Ile Tyr Glu Arg Glu Ala Pro Glu Gly 100 105 110 Thr Val Lys Asp Ile Lys Glu Gln Glu Val Tyr Met Gly Glu Ile Leu 115 120 125 Met Thr Asp Asn Gly Thr Phe Val Ile Asn Gly Thr Glu Arg Val Ile 130 135 140 Val Ser Gln Leu His Arg Ser Pro Gly Val Phe Phe Asp Ser Asp Lys 145 150 155 160 Gly Lys Thr His Ser Ser Gly Lys Val Leu Tyr Asn Ala Arg Ile Ile 165 170 175 Pro Tyr Arg Gly Ser Trp Leu Asp Phe Glu Phe Asp Pro Lys Asp Asn

180 185 190 Leu Phe Val Arg Ile Asp Arg Arg Arg Lys Leu Pro Ala Thr Ile Ile 195 200 205 Leu Arg Ala Leu Asn Tyr Thr Thr Glu Gln Ile Leu Asp Leu Phe Phe 210 215 220 Glu Lys Val Ile Phe Glu Ile Arg Asp Asn Lys Leu Gln Met Glu Leu 225 230 235 240 Val Pro Glu Arg Leu Arg Gly Glu Thr Ala Ser Phe Asp Ile Ala Asn 245 250 255 Gly Lys Val Tyr Val Glu Lys Gly Arg Arg Ile Thr Ala Arg His Ile 260 265 270 Arg Gln Leu Glu Lys Asp Asp Val Lys Leu Ile Glu Val Pro Val Glu 275 280 285 Tyr Ile Ala Gly Lys Val Val Ala Lys Asp Tyr Ile Asp Glu Ser Thr 290 295 300 Gly Glu Leu Ile Cys Ala Ala Asn Met Glu Leu Ser Leu Asp Leu Leu 305 310 315 320 Ala Lys Leu Ser Gln Ser Gly His Lys Arg Ile Glu Thr Leu Phe Thr 325 330 335 Asn Asp Leu Asp His Gly Pro Tyr Ile Ser Glu Thr Leu Arg Val Asp 340 345 350 Pro Thr Asn Asp Arg Leu Ser Ala Leu Val Glu Ile Tyr Arg Met Met 355 360 365 Arg Pro Gly Glu Pro Pro Thr Arg Glu Ala Ala Glu Ser Phe Glu Asn 370 375 380 Leu Phe Phe Ser Glu Asp Arg Tyr Asp Leu Ser Ala Val Gly Arg Met 385 390 395 400 Lys Phe Asn Arg Ser Leu Leu Arg Glu Glu Ile Glu Gly Ser Gly Ile 405 410 415 Leu Ser Lys Asp Asp Ile Ile Asp Val Met Lys Lys Leu Ile Asp Ile 420 425 430 Arg Asn Gly Lys Gly Glu Val Asp Asp Ile Asp His Leu Gly Asn Arg 435 440 445 Arg Ile Arg Ser Val Gly Glu Met Ala Glu Asn Gln Phe Arg Val Gly 450 455 460 Leu Val Arg Val Glu Arg Ala Val Lys Glu Arg Leu Ser Leu Gly Asp 465 470 475 480 Leu Asp Thr Leu Met Pro Gln Asp Met Ile Asn Ala Lys Pro Ile Ser 485 490 495 Ala Ala Val Lys Glu Phe Phe Gly Ser Ser Gln Leu Gln Phe Met Asp 500 505 510 Gln Asn Asn Pro Leu Ser Glu Ile Thr His Lys Arg Arg Ile Ser Ala 515 520 525 Leu Gly Pro Gly Gly Leu Thr Arg Glu Arg Ala Gly Phe Glu Val Arg 530 535 540 Asp Val His Pro Thr His Tyr Gly Arg Val Cys Pro Ile Glu Thr Pro 545 550 555 560 Glu Gly Pro Asn Ile Gly Leu Ile Asn Ser Leu Ser Val Tyr Ala Gln 565 570 575 Thr Asn Glu Tyr Gly Phe Leu Glu Thr Pro Tyr Arg Lys Val Thr Asp 580 585 590 Gly Val Val Thr Asp Glu Ile His Tyr Leu Ser Ala Ile Glu Glu Gly 595 600 605 Asn Tyr Val Ile Ala Gln Ala Asn Ser Asn Leu Asp Glu Glu Gly His 610 615 620 Phe Val Glu Asp Leu Val Thr Cys Arg Ser Lys Glu Ser Ser Leu Phe 625 630 635 640 Ser Arg Asp Gln Val Asp Tyr Met Asp Val Ser Thr Gln Gln Val Val 645 650 655 Ser Val Gly Ala Ser Leu Ile Pro Phe Leu Glu His Asp Asp Ala Asn 660 665 670 Arg Ala Leu Met Gly Ala Asn Met Gln Arg Gln Ala Val Pro Thr Leu 675 680 685 Arg Ala Asp Lys Pro Leu Val Gly Thr Gly Met Glu Arg Ala Val Ala 690 695 700 Val Asp Ser Gly Val Thr Ala Val Ala Lys Arg Gly Gly Val Val Gln 705 710 715 720 Tyr Val Asp Ala Ser Arg Ile Val Ile Lys Val Asn Glu Asp Glu Met 725 730 735 Tyr Pro Gly Glu Ala Gly Ile Asp Ile Tyr Asn Leu Thr Lys Tyr Thr 740 745 750 Arg Ser Asn Gln Asn Thr Cys Ile Asn Gln Pro Cys Val Ser Leu Gly 755 760 765 Glu Pro Val Glu Arg Gly Asp Val Leu Ala Asp Gly Pro Ser Thr Asp 770 775 780 Leu Gly Glu Leu Ala Leu Gly Gln Asn Met Arg Val Ala Phe Met Pro 785 790 795 800 Trp Asn Gly Tyr Asn Phe Glu Asp Ser Ile Leu Val Ser Glu Arg Val 805 810 815 Val Gln Glu Asp Arg Phe Thr Thr Ile His Ile Gln Glu Leu Ala Cys 820 825 830 Val Ser Arg Asp Thr Lys Leu Gly Pro Glu Glu Ile Thr Ala Asp Ile 835 840 845 Pro Asn Val Gly Glu Ala Ala Leu Ser Lys Leu Asp Glu Ser Gly Ile 850 855 860 Val Tyr Ile Gly Ala Glu Val Thr Gly Gly Asp Ile Leu Val Gly Lys 865 870 875 880 Val Thr Pro Lys Gly Glu Thr Gln Leu Pro Glu Glu Lys Leu Leu Arg 885 890 895 Ala Ile Phe Gly Glu Lys Ala Ser Asp Val Lys Asp Ser Ser Leu Arg 900 905 910 Val Pro Asn Gly Val Ser Gly Thr Val Ile Asp Val Gln Val Phe Thr 915 920 925 Arg Asp Gly Val Glu Lys Asp Lys Arg Ala Leu Glu Ile Glu Glu Met 930 935 940 Gln Leu Lys Gln Ala Lys Lys Asp Leu Ser Glu Glu Leu Gln Ile Leu 945 950 955 960 Glu Ala Gly Leu Phe Ser Arg Ile Arg Ala Val Leu Val Ala Gly Gly 965 970 975 Val Glu Ala Glu Lys Leu Asp Lys Leu Pro Arg Asp Arg Trp Leu Glu 980 985 990 Leu Gly Leu Thr Asp Glu Glu Lys Gln Asn Gln Leu Glu Gln Leu Ala 995 1000 1005 Glu Gln Tyr Asp Glu Leu Lys His Phe Glu Lys Lys Leu Glu Ala 1010 1015 1020 Lys Arg Arg Lys Ile Thr Gln Gly Asp Asp Leu Ala Pro Gly Val 1025 1030 1035 Leu Lys Ile Val Lys Val Tyr Leu Ala Val Lys Arg Arg Ile Gln 1040 1045 1050 Pro Gly Asp Lys Met Ala Gly Arg His Gly Asn Lys Gly Val Ile 1055 1060 1065 Ser Lys Ile Asn Pro Ile Glu Asp Met Pro Tyr Asp Glu Asn Gly 1070 1075 1080 Thr Pro Val Asp Ile Val Leu Asn Pro Leu Gly Val Pro Ser Arg 1085 1090 1095 Met Asn Ile Gly Gln Ile Leu Glu Thr His Leu Gly Met Ala Ala 1100 1105 1110 Lys Gly Ile Gly Asp Lys Ile Asn Ala Met Leu Lys Gln Gln Gln 1115 1120 1125 Glu Val Ala Lys Leu Arg Glu Phe Ile Gln Arg Ala Tyr Asp Leu 1130 1135 1140 Ala Asp Val Arg Gln Lys Val Asp Leu Ser Thr Phe Ser Asp Glu 1145 1150 1155 Glu Val Met Arg Leu Ala Glu Asn Leu Arg Lys Gly Met Pro Ile 1160 1165 1170 Ala Thr Pro Val Phe Asp Gly Ala Lys Glu Ala Glu Ile Lys Glu 1175 1180 1185 Leu Leu Lys Leu Gly Asp Leu Pro Thr Ser Gly Gln Ile Arg Leu 1190 1195 1200 Tyr Asp Gly Arg Thr Gly Glu Gln Phe Glu Arg Pro Val Thr Val 1205 1210 1215 Gly Tyr Met Tyr Met Leu Lys Leu Asn His Leu Val Asp Asp Lys 1220 1225 1230 Met His Ala Arg Ser Thr Gly Ser Tyr Ser Leu Val Thr Gln Gln 1235 1240 1245 Pro Leu Gly Gly Lys Ala Gln Phe Gly Gly Gln Arg Phe Gly Glu 1250 1255 1260 Met Glu Val Trp Ala Leu Glu Tyr Gly Ala Ala Tyr Thr Leu Gln 1265 1270 1275 Glu Met Leu Thr Val Lys Ser Asp Asp Val Asn Gly Arg Thr Lys 1280 1285 1290 Met Tyr Lys Asn Ile Val Asp Gly Asn His Gln Met Glu Pro Gly 1295 1300 1305 Met Pro Glu Ser Phe Asn Val Leu Leu Lys Glu Ile Arg Ser Leu 1310 1315 1320 Gly Ile Asn Ile Glu Leu Glu Asp Glu 1325 1330 401407PRTEscherichia coli 40Met Lys Asp Leu Leu Lys Phe Leu Lys Ala Gln Thr Lys Thr Glu Glu 1 5 10 15 Phe Asp Ala Ile Lys Ile Ala Leu Ala Ser Pro Asp Met Ile Arg Ser 20 25 30 Trp Ser Phe Gly Glu Val Lys Lys Pro Glu Thr Ile Asn Tyr Arg Thr 35 40 45 Phe Lys Pro Glu Arg Asp Gly Leu Phe Cys Ala Arg Ile Phe Gly Pro 50 55 60 Val Lys Asp Tyr Glu Cys Leu Cys Gly Lys Tyr Lys Arg Leu Lys His 65 70 75 80 Arg Gly Val Ile Cys Glu Lys Cys Gly Val Glu Val Thr Gln Thr Lys 85 90 95 Val Arg Arg Glu Arg Met Gly His Ile Glu Leu Ala Ser Pro Thr Ala 100 105 110 His Ile Trp Phe Leu Lys Ser Leu Pro Ser Arg Ile Gly Leu Leu Leu 115 120 125 Asp Met Pro Leu Arg Asp Ile Glu Arg Val Leu Tyr Phe Glu Ser Tyr 130 135 140 Val Val Ile Glu Gly Gly Met Thr Asn Leu Glu Arg Gln Gln Ile Leu 145 150 155 160 Thr Glu Glu Gln Tyr Leu Asp Ala Leu Glu Glu Phe Gly Asp Glu Phe 165 170 175 Asp Ala Lys Met Gly Ala Glu Ala Ile Gln Ala Leu Leu Lys Ser Met 180 185 190 Asp Leu Glu Gln Glu Cys Glu Gln Leu Arg Glu Glu Leu Asn Glu Thr 195 200 205 Asn Ser Glu Thr Lys Arg Lys Lys Leu Thr Lys Arg Ile Lys Leu Leu 210 215 220 Glu Ala Phe Val Gln Ser Gly Asn Lys Pro Glu Trp Met Ile Leu Thr 225 230 235 240 Val Leu Pro Val Leu Pro Pro Asp Leu Arg Pro Leu Val Pro Leu Asp 245 250 255 Gly Gly Arg Phe Ala Thr Ser Asp Leu Asn Asp Leu Tyr Arg Arg Val 260 265 270 Ile Asn Arg Asn Asn Arg Leu Lys Arg Leu Leu Asp Leu Ala Ala Pro 275 280 285 Asp Ile Ile Val Arg Asn Glu Lys Arg Met Leu Gln Glu Ala Val Asp 290 295 300 Ala Leu Leu Asp Asn Gly Arg Arg Gly Arg Ala Ile Thr Gly Ser Asn 305 310 315 320 Lys Arg Pro Leu Lys Ser Leu Ala Asp Met Ile Lys Gly Lys Gln Gly 325 330 335 Arg Phe Arg Gln Asn Leu Leu Gly Lys Arg Val Asp Tyr Ser Gly Arg 340 345 350 Ser Val Ile Thr Val Gly Pro Tyr Leu Arg Leu His Gln Cys Gly Leu 355 360 365 Pro Lys Lys Met Ala Leu Glu Leu Phe Lys Pro Phe Ile Tyr Gly Lys 370 375 380 Leu Glu Leu Arg Gly Leu Ala Thr Thr Ile Lys Ala Ala Lys Lys Met 385 390 395 400 Val Glu Arg Glu Glu Ala Val Val Trp Asp Ile Leu Asp Glu Val Ile 405 410 415 Arg Glu His Pro Val Leu Leu Asn Arg Ala Pro Thr Leu His Arg Leu 420 425 430 Gly Ile Gln Ala Phe Glu Pro Val Leu Ile Glu Gly Lys Ala Ile Gln 435 440 445 Leu His Pro Leu Val Cys Ala Ala Tyr Asn Ala Asp Phe Asp Gly Asp 450 455 460 Gln Met Ala Val His Val Pro Leu Thr Leu Glu Ala Gln Leu Glu Ala 465 470 475 480 Arg Ala Leu Met Met Ser Thr Asn Asn Ile Leu Ser Pro Ala Asn Gly 485 490 495 Glu Pro Ile Ile Val Pro Ser Gln Asp Val Val Leu Gly Leu Tyr Tyr 500 505 510 Met Thr Arg Asp Cys Val Asn Ala Lys Gly Glu Gly Met Val Leu Thr 515 520 525 Gly Pro Lys Glu Ala Glu Arg Leu Tyr Arg Ser Gly Leu Ala Ser Leu 530 535 540 His Ala Arg Val Lys Val Arg Ile Thr Glu Tyr Glu Lys Asp Ala Asn 545 550 555 560 Gly Glu Leu Val Ala Lys Thr Ser Leu Lys Asp Thr Thr Val Gly Arg 565 570 575 Ala Ile Leu Trp Met Ile Val Pro Lys Gly Leu Pro Tyr Ser Ile Val 580 585 590 Asn Gln Ala Leu Gly Lys Lys Ala Ile Ser Lys Met Leu Asn Thr Cys 595 600 605 Tyr Arg Ile Leu Gly Leu Lys Pro Thr Val Ile Phe Ala Asp Gln Ile 610 615 620 Met Tyr Thr Gly Phe Ala Tyr Ala Ala Arg Ser Gly Ala Ser Val Gly 625 630 635 640 Ile Asp Asp Met Val Ile Pro Glu Lys Lys His Glu Ile Ile Ser Glu 645 650 655 Ala Glu Ala Glu Val Ala Glu Ile Gln Glu Gln Phe Gln Ser Gly Leu 660 665 670 Val Thr Ala Gly Glu Arg Tyr Asn Lys Val Ile Asp Ile Trp Ala Ala 675 680 685 Ala Asn Asp Arg Val Ser Lys Ala Met Met Asp Asn Leu Gln Thr Glu 690 695 700 Thr Val Ile Asn Arg Asp Gly Gln Glu Glu Lys Gln Val Ser Phe Asn 705 710 715 720 Ser Ile Tyr Met Met Ala Asp Ser Gly Ala Arg Gly Ser Ala Ala Gln 725 730 735 Ile Arg Gln Leu Ala Gly Met Arg Gly Leu Met Ala Lys Pro Asp Gly 740 745 750 Ser Ile Ile Glu Thr Pro Ile Thr Ala Asn Phe Arg Glu Gly Leu Asn 755 760 765 Val Leu Gln Tyr Phe Ile Ser Thr His Gly Ala Arg Lys Gly Leu Ala 770 775 780 Asp Thr Ala Leu Lys Thr Ala Asn Ser Gly Tyr Leu Thr Arg Arg Leu 785 790 795 800 Val Asp Val Ala Gln Asp Leu Val Val Thr Glu Asp Asp Cys Gly Thr 805 810 815 His Glu Gly Ile Met Met Thr Pro Val Ile Glu Gly Gly Asp Val Lys 820 825 830 Glu Pro Leu Arg Asp Arg Val Leu Gly Arg Val Thr Ala Glu Asp Val 835 840 845 Leu Lys Pro Gly Thr Ala Asp Ile Leu Val Pro Arg Asn Thr Leu Leu 850 855 860 His Glu Gln Trp Cys Asp Leu Leu Glu Glu Asn Ser Val Asp Ala Val 865 870 875 880 Lys Val Arg Ser Val Val Ser Cys Asp Thr Asp Phe Gly Val Cys Ala 885 890 895 His Cys Tyr Gly Arg Asp Leu Ala Arg Gly His Ile Ile Asn Lys Gly 900 905 910 Glu Ala Ile Gly Val Ile Ala Ala Gln Ser Ile Gly Glu Pro Gly Thr 915 920 925 Gln Leu Thr Met Arg Thr Phe His Ile Gly Gly Ala Ala Ser Arg Ala 930 935 940 Ala Ala Glu Ser Ser Ile Gln Val Lys Asn Lys Gly Ser Ile Lys Leu 945 950 955 960 Ser Asn Val Lys Ser Val Val Asn Ser Ser Gly Lys Leu Val Ile Thr 965 970 975 Ser Arg Asn Thr Glu Leu Lys Leu Ile Asp Glu Phe Gly Arg Thr Lys 980 985 990 Glu Ser Tyr Lys Val Pro Tyr Gly Ala Val Leu Ala Lys Gly Asp Gly 995 1000 1005 Glu Gln Val Ala Gly Gly Glu Thr Val Ala Asn Trp Asp Pro His 1010 1015 1020 Thr Met Pro Val Ile Thr Glu Val Ser Gly Phe Val Arg Phe Thr 1025 1030 1035 Asp Met Ile Asp Gly Gln Thr Ile Thr Arg Gln Thr Asp Glu Leu 1040 1045 1050 Thr Gly Leu Ser Ser Leu Val Val Leu Asp Ser Ala Glu Arg Thr 1055 1060 1065 Ala Gly Gly Lys Asp Leu Arg Pro Ala Leu Lys Ile Val Asp Ala 1070 1075 1080 Gln Gly Asn Asp Val Leu Ile Pro Gly Thr Asp Met Pro Ala Gln 1085 1090 1095 Tyr Phe Leu Pro Gly Lys Ala Ile Val Gln Leu Glu Asp Gly Val 1100 1105 1110 Gln Ile Ser Ser Gly Asp Thr Leu Ala Arg Ile Pro Gln Glu Ser 1115 1120 1125 Gly Gly

Thr Lys Asp Ile Thr Gly Gly Leu Pro Arg Val Ala Asp 1130 1135 1140 Leu Phe Glu Ala Arg Arg Pro Lys Glu Pro Ala Ile Leu Ala Glu 1145 1150 1155 Ile Ser Gly Ile Val Ser Phe Gly Lys Glu Thr Lys Gly Lys Arg 1160 1165 1170 Arg Leu Val Ile Thr Pro Val Asp Gly Ser Asp Pro Tyr Glu Glu 1175 1180 1185 Met Ile Pro Lys Trp Arg Gln Leu Asn Val Phe Glu Gly Glu Arg 1190 1195 1200 Val Glu Arg Gly Asp Val Ile Ser Asp Gly Pro Glu Ala Pro His 1205 1210 1215 Asp Ile Leu Arg Leu Arg Gly Val His Ala Val Thr Arg Tyr Ile 1220 1225 1230 Val Asn Glu Val Gln Asp Val Tyr Arg Leu Gln Gly Val Lys Ile 1235 1240 1245 Asn Asp Lys His Ile Glu Val Ile Val Arg Gln Met Leu Arg Lys 1250 1255 1260 Ala Thr Ile Val Asn Ala Gly Ser Ser Asp Phe Leu Glu Gly Glu 1265 1270 1275 Gln Val Glu Tyr Ser Arg Val Lys Ile Ala Asn Arg Glu Leu Glu 1280 1285 1290 Ala Asn Gly Lys Val Gly Ala Thr Tyr Ser Arg Asp Leu Leu Gly 1295 1300 1305 Ile Thr Lys Ala Ser Leu Ala Thr Glu Ser Phe Ile Ser Ala Ala 1310 1315 1320 Ser Phe Gln Glu Thr Thr Arg Val Leu Thr Glu Ala Ala Val Ala 1325 1330 1335 Gly Lys Arg Asp Glu Leu Arg Gly Leu Lys Glu Asn Val Ile Val 1340 1345 1350 Gly Arg Leu Ile Pro Ala Gly Thr Gly Tyr Ala Tyr His Gln Asp 1355 1360 1365 Arg Met Arg Arg Arg Ala Ala Gly Glu Ala Pro Ala Ala Pro Gln 1370 1375 1380 Val Thr Ala Glu Asp Ala Ser Ala Ser Leu Ala Glu Leu Leu Asn 1385 1390 1395 Ala Gly Leu Gly Gly Ser Asp Asn Glu 1400 1405 41327PRTCitrobacter koseri 41Met Gln Gly Ser Val Thr Glu Phe Leu Lys Pro Arg Leu Val Asp Ile 1 5 10 15 Glu Gln Val Ser Ser Thr His Ala Lys Val Thr Leu Glu Pro Leu Glu 20 25 30 Arg Gly Phe Gly His Thr Leu Gly Asn Ala Leu Arg Arg Ile Leu Leu 35 40 45 Ser Ser Met Pro Gly Cys Ala Val Thr Glu Val Glu Ile Asp Gly Val 50 55 60 Leu His Glu Tyr Ser Thr Lys Glu Gly Val Gln Glu Asp Ile Leu Glu 65 70 75 80 Ile Leu Leu Asn Leu Lys Gly Leu Ala Val Arg Val Gln Gly Lys Asp 85 90 95 Glu Val Ile Leu Thr Leu Asn Lys Ser Gly Ile Gly Pro Val Thr Ala 100 105 110 Ala Asp Ile Thr His Asp Gly Asp Val Glu Ile Val Lys Pro Gln Val 115 120 125 Ile Cys His Leu Thr Asp Glu Asn Ala Ser Ile Ser Met Arg Ile Lys 130 135 140 Val Gln Arg Gly Arg Gly Tyr Val Pro Ala Ser Thr Arg Ile His Ser 145 150 155 160 Glu Glu Asp Glu Arg Pro Ile Gly Arg Leu Leu Val Asp Ala Cys Tyr 165 170 175 Ser Pro Val Glu Arg Ile Ala Tyr Asn Val Glu Ala Ala Arg Val Glu 180 185 190 Gln Arg Thr Asp Leu Asp Lys Leu Val Ile Glu Met Glu Thr Asn Gly 195 200 205 Thr Ile Asp Pro Glu Glu Ala Ile Arg Arg Ala Ala Thr Ile Leu Ala 210 215 220 Glu Gln Leu Glu Ala Phe Val Asp Leu Arg Asp Val Arg Gln Pro Glu 225 230 235 240 Val Lys Glu Glu Lys Pro Glu Phe Asp Pro Ile Leu Leu Arg Val Asp 245 250 255 Asp Leu Glu Leu Thr Val Arg Ser Ala Asn Cys Leu Lys Ala Glu Ala 260 265 270 Ile His Tyr Ile Gly Asp Leu Val Gln Arg Thr Glu Val Glu Leu Leu 275 280 285 Lys Thr Pro Asn Leu Gly Lys Lys Ser Leu Thr Glu Ile Lys Asp Val 290 295 300 Leu Ala Ser Arg Gly Leu Ser Leu Gly Met Arg Leu Glu Asn Trp Pro 305 310 315 320 Pro Ala Ser Ile Ala Asp Glu 325 42402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 42gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt attactgtgc gagagggacc 300gattacacta ttgacgacca ggggatcckt tatcaaggtt cggggacctt ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag nn 40243402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 43gaggttcagc tggtggagtc tggggcaaat gttgtacggc cgggggggtc cctgagactc 60tcctgtaaag cgtccggatt catctttgaa aattttggtt ttagttgggt ccgccaggct 120ccagggaagg ggcttcagtg ggtcgctggt cttaattgga atggtggtga cacacgttat 180gcagactctg tgaagggccg attcagaatg tccagagaca actccaggaa ttttgtgtat 240ttggacatgg ataaagtggg agtcgacgac acggccttct attactgtgc gagagggacc 300gattacacta ttgacgacgc ggggatccat taccaaggtt cggggacctt ctggtacttc 360gatctctggg gccgtggcac cctggtcagt gtctcttcag nn 40244402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 44gaggttcagc tggtggagtc tgggggaagt gtggtgcggc cgggggggtc cctgagactc 60tcctgtagag cgtccggatt catctttgag aactatggcc tgacttgggt ccgccaagtt 120ccagggaaag ggctacattg ggtctccggg atgaattgga atggtggtga cacgcgttat 180gcagactctg tgaggggccg atttagcatg tccagagaca acagcaacaa catcgcatat 240ctgcaaatga ataatctgag agtggaggac acggccttrt attactgcgc gagagggacc 300gattacacga tagacgacca gggaagatkt tatcaaggat cggggacctt ctggtacttc 360gatttttggg gccgtggcac actggtcact gtctcttcag nn 40245402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 45gaggtgcagc tggtggagtc tgggggaggt gtggtgcggc cgggggggtc cctgagactc 60tcctgtgcag cgtccggatt catttttgag aactacggct tgacttgggt ccgccaagtt 120ccagggaaag ggctgcattg ggtctccggt atgaattgga atggtggtga cacgcgttat 180gcagactctg tgaggggccg attcagcatg tccagagaca acagcaataa tatcgcatat 240ctgcaaatga aaaatctgag agtcgacgac acggccttrt attactgtgc gagagggacc 300gattacacga tagacgacca gggaatttkt tataaaggtt cggggacctt ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcttcag nn 40246402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 46gaggtkcagc tggtggagtc tgggggaggt ctcatacggc cgggggggtc cctgagactc 60tcctgtaaag gctccggttt catctttgag aattttggct tcggctgggt ccgccaaggt 120ccagggaagg ggctggagtg ggtgtctggc actaattgga atggaggtga ctcacgttat 180ggagactctg tgaagggccg attcacaatc tccagagaca acagcaacaa tttcgtctac 240ctgcaaatga acagtctgag acccgaggac acggccatrt attattgtgc gagagggacc 300gattacacta ttgacgatca ggggatcckt tatcaaggtt cggggacttt ctggtacttc 360gatgtctggg gccgcggcac cctggtcacg gtctcctcag nn 40247402DNAHomo sapiensmodified_base(401)..(402)a, c, t, g, unknown or other 47gaggtkcagc tggtggagtc tgggggaggt ctcatacggc cgggggggtc cctgagactc 60tcctgtaaag gctccggttt catctttgag aattttggct tcggctgggt ccgccaaggt 120ccagggaagg ggctggagtg ggtgtctggc actaattgga atggaggtga ctcacgttat 180ggagactctg tgaagggccg attcacaatc tccagagaca acagcaacaa tttcgtctac 240ctgcaaatga acagtctgag acccgaggac acggccatrt attattgtgc gagagggacc 300gattacacta ttgacgatca ggggatcckt tatcaaggtt cggggacttt ctggtacttc 360gatgtctggg gccgcggcac cctggtcacg gtctcctcag nn 40248322DNAHomo sapiens 48gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 32249322DNAHomo sapiens 49gaaattgtgt tgacacagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca caatgtccac cccaaatatt tcgcctggta ccagcagaag 120cctggccagt ctccccgact cctcatctat ggtgggtcca ccagggccgc tggcattcca 180ggcaagttca gcggcagtgg gtctgggacc gacttcactc tcaccatcag tcgagtggac 240cctgaagatt ttgcagttta ttactgtcag cagtatggtg gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 32250322DNAHomo sapiens 50gaaattgtgt tgacgcagtc tccagccacc ctgtctgtgt ctccggggga gagagccacc 60ctctcctgca gggccagtca gaatgtccac cccagatatt tcgcctggta tcaacaaaaa 120cgtggccagt ctcccaggct cctcatccat agtggatcca ccagggccgc tggcatcgca 180gacaggttca gtggcggtgg gtctggaatg cacttcactc tcaccatcac cagagtggag 240cctgaagatt ttgcagtcta tttctgtcaa caatacggtg gttctcccta cacgttcggc 300caggggacca gggtggaact ca 32251322DNAHomo sapiens 51gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt ctccggggga aagagccacc 60ctctcctgca gggccagtca gagtgtccac cccaaatatt tcgcctggta ccagcagaaa 120cctggccagt ctcccaggct cctcatctat agtggatcca ctagggccgc tggcatcgca 180gacaggttca gtggcggtgg gtctggaata cacttcactc tcaccatcac cagagtggag 240cctgaagatt ttgcagtgta tttctgtcaa caatacggtg gttcccccta cacgttcggc 300caggggacca aggtggaact ca 32252322DNAHomo sapiens 52gaaattgtgt tgacrcagtc tccagacacc ctgtctttgt ccccagggga gagagccacc 60ctctcatgca gggccagtca gagtgttcac agcagatact ttgcctggta ccagcataaa 120cctggccagc ctcccagact cctcatctat ggtgggtcca ccagggccac tggcatccct 180aatagattca gtgccggcgg gtctgggaca cagttcactc tcaccgtcaa cagactggag 240gctgaagatt ttgcggtata ttactgtcag cagtatggtc gctccccgta cacgttcggc 300caagggacca aggtggagat ca 32253324DNAHomo sapiens 53gacatccagw tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaagcca 120ggtaaagccc ataagctcct catctatgct gcatctagtt tacaaagtgg ggtcccatca 180cggttcagcg gcagtgggtc tggcacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacaa gattacagtt acccgtatac ttttggccag 300gggaccaacc tggagatcaa gcga 32454400DNAHomo sapiens 54gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt attactgtgc gagagggacc 300gattacacta ttgacgacca ggggatcctt tatcaaggtt cggggacctt ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40055322DNAHomo sapiens 55gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 32256400DNAHomo sapiens 56gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt attactgtgc gagagggacc 300gattacacta ttgacgacca ggggatccgt tatcaaggtt cggggacctt ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40057322DNAHomo sapiens 57gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 32258400DNAHomo sapiens 58gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacta ttgacgacgc ggggatccat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40059321DNAHomo sapiens 59aaattgtgtt gacgcagtct ccaggcaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtgtat tactgtcagc agtatggtgg ttccccctac acgttcggcc 300aagggaccaa ggtggaaatc a 32160400DNAHomo sapiens 60gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacga tagacgacca gggaagatat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40061321DNAHomo sapiens 61aaattgtgtt gacgcagtct ccagccaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg atgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtctat tactgtcagc aatacggtgg ttctccctac acttttggcc 300aggggaccaa gctggagatc a 32162400DNAHomo sapiens 62gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacga tagacgacca gggaagatat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40063321DNAHomo sapiens 63aaattgtgtt gacgcagtct ccagccaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg atgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtctat tactgtcagc aatacggtgg ttccccctac acgttcggcc 300aagggaccaa ggtggaaatc a 32164400DNAHomo sapiens 64gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacga tagacgacca gggaatttat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40065321DNAHomo sapiens 65aaattgtgtt gacgcagtct ccaggcaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtgtat tactgtcagc agtatggtgg ttccccctac acttttggcc 300aggggaccaa gctggagatc a 32166400DNAHomo sapiens 66gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacga tagacgacca gggaatttat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40067321DNAHomo sapiens 67aaattgtgtt gacgcagtct ccaggcaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtgtat tactgtcagc agtatggtgg ttccccctac acgttcggcc 300aagggaccaa ggtggaaatc a 32168400DNAHomo sapiens 68gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc

60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt atcactgtgc gagagggacc 300gattacacga tagacgacca gggaatttat tactatggtt cggggaccta ctggtacttc 360gatctctggg gccgtggcac cctggtcact gtctcctcag 40069321DNAHomo sapiens 69aaattgtgtt gacgcagtct ccagccaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg atgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtctat tactgtcagc aatacggtgg ttctccctac acttttggcc 300aggggaccaa gctggagatc a 32170280DNAHomo sapiens 70gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt 2807150DNAHomo sapiens 71ctggtacttc gatctctggg gccgtggcac cctggtcact gtctcctcag 5072321DNAHomo sapiens 72aaattgtgtt gacgcagtct ccagccaccc tgtctttgtc tccaggggaa agagccaccc 60tctcctgcag ggccagtcag agtgttagca gcagctactt agcctggtac cagcagaaac 120ctggccaggc tcccaggctc ctcatctatg atgcatccag cagggccact ggcatcccag 180acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agactggagc 240ctgaagattt tgcagtctat tactgtcagc aatacggtgg ttccccctac acgttcggcc 300aagggaccaa ggtggaaatc a 32173280DNAHomo sapiens 73gaggttcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt 2807450DNAHomo sapiens 74ctggtacttc gatctctggg gccgtggcac cctggtcact gtctcctcag 5075322DNAHomo sapiens 75gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 32276280DNAHomo sapiens 76gaggtgcagc tggtggagtc tgggggaggt gtggtacggc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttgat gattatggca tgagctgggt ccgccaagct 120ccagggaagg ggctggagtg ggtctctggt attaattgga atggtggtag cacaggttat 180gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agccgaggac acggccttrt 2807750DNAHomo sapiens 77ctggtacttc gatctctggg gccgtggcac cctggtcact gtctcctcag 5078322DNAHomo sapiens 78gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctccccgta cacgttcggc 300caagggacca aggtggaaat ca 3227932PRTHerpes simplex virus 79Lys Tyr Ala Leu Val Asp Ala Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asp Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro 20 25 30 8032PRTHerpes simplex virus 80Lys Tyr Ala Leu Val Asp Pro Ser Leu Lys Met Ala Asp Pro Asn Arg 1 5 10 15 Phe Arg Gly Lys Asn Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro 20 25 30 819PRTHomo sapiens 81Arg Gly Thr Asp Tyr Thr Ile Asp Asp 1 5 826PRTHomo sapiens 82Thr Asp Tyr Thr Ile Asp 1 5 8311PRTHomo sapiens 83Asp Tyr Arg Asn Gly Tyr Asn Tyr Tyr Asp Phe 1 5 10 849PRTHomo sapiens 84Ala Phe Ile Lys Tyr Asp Gly Ser Glu 1 5 859PRTHomo sapiens 85Tyr Tyr Asp Phe Tyr Asp Gly Tyr Tyr 1 5 869PRTHomo sapiens 86Glu Asp Gly Asp Tyr Leu Ser Asp Pro 1 5 8710PRTHomo sapiens 87Asp Gly Asp Tyr Leu Ser Asp Pro Phe Tyr 1 5 10 8817PRTHomo sapiens 88Asp Gly Asp Tyr Leu Ser Asp Pro Phe Tyr Tyr Asn His Gly Met Asp 1 5 10 15 Val 8912PRTHomo sapiens 89Pro Tyr Pro Asn Asp Tyr Asn Asp Tyr Ala Pro Glu 1 5 10 9010PRTHomo sapiens 90Asn Asp Tyr Asn Asp Tyr Ala Pro Glu Glu 1 5 10 918PRTHomo sapiens 91Asn Asp Tyr Asn Asp Tyr Ala Pro 1 5 929PRTHomo sapiens 92Asp Tyr Asn Asp Tyr Ala Pro Glu Glu 1 5 937PRTHomo sapiens 93Asp Tyr Ala Pro Glu Glu Gly 1 5 949PRTHomo sapiens 94Ala Ala Gly Asp Tyr Ala Asp Tyr Asp 1 5 959PRTHomo sapiens 95Asp Tyr Ala Asp Tyr Asp Gly Gly Tyr 1 5 969PRTHomo sapiens 96Tyr Asp Gly Gly Tyr Tyr Tyr Asp Met 1 5

Claims

1. A method of inducing the production in a subject of broadly neutralizing antibodies against HIV-1 comprising: i) administering to said subject a non-HIV-1 antigen that binds to a germline B cell receptor, said non-HIV-1 antigen being administered in an amount and under conditions such that intermediate clones of B cells are produced that secrete antibodies that cross-react with HIV-1 Env, and ii) administering to said subject an HIV-1 antigen in an amount and under conditions such that naive B cells or said intermediate clones of B cells are produced that secrete said broadly neutralizing anti-HIV-1 antibodies.

2. The method according to claim 1 wherein said subject is a human.

3. The method according to claim 1 wherein said non-HIV-1 antigen is a lipid.

4. The method according to claim 3 wherein said lipid is cardiolipin, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphotidylinositol, sphingomyelin, or derivative thereof.

5. The method according to claim 4 wherein said lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), or dioleoyl phosphatidylethanolamine (DOPE).

6. The method according to claim 3 wherein said lipid is a hexagonal II phase of a phospholipid.

7. The method according to claim 1 wherein said non-HIV-1 antigen is phycoerythrin (PE), C-phycocyanin (C-PC), apoferritin, or anerobic or aerobic gut flora or component thereof.

8. The method according to claim 1 wherein said non-HIV antigen comprises the a subunit of RNA polymerase core protein of a bacteria or eukaryote.

9. The method according to claim 1 wherein said non-HIV antigen is kynureninase (KYNU) or antigenic fragment thereof.

10. The method according to claim 9 wherein said KYNU is recombinant KYNU expressed in CHO or 293T cells, or antigenic fragment thereof.

11. The method according to claim 1 further comprising administering an adjuvant.

12. The method according to claim 11 wherein said adjuvant is squalene based adjuvant, a TRL agonist, an oligonucleotide (oCpGs) or R848.

13. The method according to claim 1 wherein said HIV-1 antigen is a membrane-proximal external region (MPER) antigen, or variant thereof.

14. The method according to claim 13 wherein said HIV-1 antigen is an immunogen shown in FIGS. 16B, 16C, 17, 18, 20, 25 or 26.

15. The method according to claim 13 wherein said variant is a MPER epitope peptide with an L669S mutation.

16. The method according to claim 1 wherein said HIV-1 antigen is a transmitted founder HIV-1 Env, or antigenic fragment thereof.

17. The method according to claim 16 wherein said fragment comprises a portion of the CD4 binding site of gp120, an MPER sequence, or a portion of gp120 comprising the V2 or V3 region of gp120.

18. The method according to claim 1 wherein the method is effected by administering to said subject a prime immunization comprising said non-HIV-1 antigen followed by one or more boosts comprising said HIV-1 antigen.

19. The method according to claim 1 wherein said non-HIV-1 antigen comprises a lipid, a component of anaerobic or aerobic gut flora bacteria, phycobiliprotein, or KYNU or fragment thereof, and said HIV-1 antigen comprises an HIV-1 Env antigen selected from the group consisting of transmitted founder Env 1086.C from Malawi, 089.0 from Malawi, 040_C9 from the U.S. and 63521 from a Clade B acute HIV-1 infected U.S. patient.

20. The method according to claim 1 wherein said non-HIV antigen or said HIV antigen comprises a protein and a DNA sequence encoding said protein is administered to said subject under conditions such that said DNA sequence is expressed and said protein is thereby produced.

21. The method according to claim 1 wherein A244gD+ envelope is administered as a prime and an envelope bound by CHO1, CHO2, CHO3, CHO4 or CHO5 is administered as a boost.

22. The method according to claim 1 wherein said non-HIV-1 antigen is present in a liposome with said HIV-1 antigen and at least one adjuvant.

23. The method according to claim 1 wherein said non-HIV-1 antigen is conjugated to said HIV-1 antigen and formulated with one or more adjuvants.

24. An antibody selected from the group consisting of CHO1, CHO2, CHO3, CHO4, and CHO5, or antigen binding fragment thereof.

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