Patent Publication Number: US-10322141-B2

Title: Compositions comprising CH848 envelopes and uses thereof

Description:
This application is a U.S. National Stage application under 35 U.S.C. § 371 of International Patent Application No. PCT/US15/23632, filed Mar. 31, 2015, which claims the benefit of and priority of U.S. Application Ser. No. 61/972,649, filed Mar. 31, 2014, the contents of which application are herein incorporated by reference in their entireties. 
     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 Jun. 15, 2017, is named 2017-06-19 239 US1 App No. 15300051 Sequence Listing and is 3,082,557 bytes in size 
    
    
     This invention was made with government support under Center for HIV/AIDS Vaccine Immunology-Immunogen Design grant UM1-A1100645 from the NIH, NIAID, Division of AIDS. The government has certain rights in the invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates in general, to a composition suitable for use in inducing anti-HIV-1 antibodies, and, in particular, to immunogenic compositions comprising envelope proteins and nucleic acids to induce cross-reactive neutralizing antibodies and increase their breadth of coverage. The invention also relates to methods of inducing such broadly neutralizing anti-HIV-1 antibodies using such compositions. 
     BACKGROUND 
     The development of a safe and effective HIV-1 vaccine is one of the highest priorities of the scientific community working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients, ART is not routinely available in developing countries. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to HIV-1 immunogens and uses thereof. In certain aspects the invention provides immunogenic compositions comprising HIV-1 envelopes and their uses in methods to induce immune response. In certain aspects, the immune responses induced are broadly neutralizing antibodies. 
     In certain embodiments, the invention provides compositions and method for induction of immune response, for example cross-reactive (broadly) neutralizing Ab induction. In certain embodiments, the methods use compositions comprising “swarms” of sequentially evolved envelope viruses that occur in the setting of bnAb generation in vivo in HIV-1 infection. 
     In certain aspects the invention provides compositions comprising a selection of HIV-1 envelopes, or nucleic acids encoding these envelopes, or a combination thereof as described herein for example but not limited to selections as described herein. In certain embodiments, these envelopes are used in immunization methods as a prime and/or boost(s). 
     In one aspect the invention provides a composition comprising nucleic acids encoding HIV-1 envelopes as described herein. In certain embodiments, the compositions contemplate nucleic acid, as DNA and/or RNA, or proteins immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and/or RNA, immunization either with nucleic acids alone or in combination with envelope protein(s). In certain embodiments, the nucleic acids and the protein are directed to the same envelope variant, as a non-limiting example gp120. In other embodiments, the nucleic acids encode one variant, as a non-limiting example gp160 or gp145, while the corresponding envelope protein is another variant, as a non-limiting example gp120, or gp140. 
     In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant. 
     In certain embodiments the induced immune response includes induction of antibodies, including but not limited to autologous and/or cross-reactive (broadly) neutralizing antibodies against HIV-1 envelope. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are known in the art and are also described herein. 
     In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acid comprising any one of the nucleic acid sequences of invention. A nucleic acid consisting essentially of any one of the nucleic acid sequences of invention. A nucleic acid consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector. 
     In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention. 
     In certain embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide instead of a nucleic acid sequence encoding the HIV-1 envelope. In certain embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide, a nucleic acid sequence encoding the HIV-1 envelope, or a combination thereof. The envelope can be a gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. The polypeptide contemplated by the invention can be a polypeptide comprising any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting essentially of any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting of any one of the polypeptides described herein. In certain embodiments, the polypeptide is recombinantly produced. In certain embodiments, the polypeptides and nucleic acids of the invention are suitable for use as an immunogen, for example to be administered in a human subject. 
     In certain aspects the invention provides a composition comprising a nucleic acid encoding HIV-1 envelope as described herein or any combination thereof, e.g.  FIG. 1  or any combination thereof, or  FIG. 2  or any combination thereof, or  FIG. 6  or any combination thereof, or  FIG. 8  or any combination thereof, or any suitable variants such as gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. 
     In certain embodiments, the nucleic acid encoding the CH848 envelope encodes a gp160 envelope. In certain embodiments, the nucleic acid encoding the CH848 envelope encodes a gp140 envelope. In certain embodiments, the nucleic acid encoding the CH848 envelope encodes a gp120 or D11gp120 envelope. 
     In certain aspects the invention provides a composition comprising any one of the polypeptides of  FIG. 1  or any combination thereof, or  FIG. 2  or any combination thereof, or  FIG. 6  or any combination thereof, or  FIG. 8  or any combination thereof, or any suitable variants such as gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. 
     In certain embodiments, the polypeptide is a gp160 envelope. In certain embodiments, the polypeptide is a gp140 envelope. In certain embodiments, the polypeptide is a gp120 envelope. In certain embodiments, the polypeptide is recombinantly expressed. 
     In certain embodiments, the nucleic acid is operably linked to a promoter inserted in an expression vector. 
     In certain aspects, the invention provides an immunogenic composition comprises any one of the envelopes described herein and further comprising an adjuvant. 
     In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering a composition comprising a suitable HIV-1 envelope from CH848, e.g. CH84 T/F, as a prime in an amount sufficient to induce an immune response. In certain embodiments, the envelope is administered as gp160. In other embodiments, the envelope is administered as gp120. In other embodiments, the envelope is administered as any suitable variant, e.g. gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. In certain embodiments, the envelope is administered as a nucleic acid, a protein, or a combination thereof. 
     In certain embodiments, the method further comprises administering a composition comprising any one of the CH848 envelopes described herein, or any combination thereof, for example, the envelope is administered as any suitable variant, e.g. gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. In certain embodiments, the envelope or combinations thereof are administered as nucleic acids, protein, or a combination thereof. 
     In certain embodiments of the methods, the envelope is administered as a protein or a nucleic acid encoding the envelope, or any combination thereof. In certain embodiments of the methods, the protein is recombinant. In certain embodiments of the methods, the nucleic acid encoding the envelope is operably linked to a promoter inserted in an expression vector. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows amino acids encoding gp160s envelopes from CH848 (SEQ ID NOs: 2 through 101 in order of appearance). d0135.27.03 represents the T/F form. 
         FIG. 2  shows amino acid sequence of gp120 envelopes from CH848. d0135.27.03 represents the T/F form. (SEQ ID NOs: 102 through 201 in order of appearance). 
         FIG. 3  shows one embodiment of optimized nucleic acid sequences encoding the gp160s of  FIG. 2 . (SEQ ID NOs: 202 through 278 in order of appearance). Coding sequence is in capital letters. 
         FIG. 4  shows CH848 TF gp160 wild-type DNA and a.a sequences, examples of gp140C and delta 11 deletion gp120 design. Coding sequence is in capital letters. Sequences are identified by the sequence identifiers listed in the Figure. 
         FIG. 5  shows DNA sequence of CH848D11gp120 constructs. (SEQ ID NOs: 287 through 386 in order of appearance). Coding sequence starts with the first ATG in capital letters. 
         FIG. 6  shows amino acid sequence of CH848D11gp120 constructs. (Coding sequence ends with **; SEQ ID NOs: 387 through 558 in order of appearance). 
         FIG. 7  shows DNA sequence of CH848gp140C constructs. (SEQ ID NOs: 559 through 658 in order of appearance). Coding sequence starts with the first ATG in capital letters. 
         FIG. 8  shows amino acids sequences of CH848gp140C constructs. (Coding sequence ends with **; SEQ ID NOs: 659 through 830 in order of appearance). 
         FIG. 9  shows that CH0848 Exhibits Broad Heterologous Neutralization. CH0848 was infected by a single, Clade C T/F virus. 
         FIG. 10  shows phylogeny of 1,223 Env single genome sequences (SGSs) from CH0848 Generated over 5 Years of Infection (this pixel and tree figure is Env only, all sequences identical to TF are omitted (˜200)). 
         FIG. 11  shows Autologous and Heterologous Plasma Neutralization in CH0848 
         FIGS. 12A and 12B  show time points for memory B-culture (upward arrow; to isolate antibodies) and sequencing (downward arrow; could be antibody and virus sequencing). 
     
    
    
     DETAILED DESCRIPTION 
     The development of a safe, highly efficacious prophylactic HIV-1 vaccine is of paramount importance for the control and prevention of HIV-1 infection. A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) (Immunol. Rev. 254: 225-244, 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not induced by current vaccines. 
     For the past 25 years, the HIV vaccine development field has used single or prime boost heterologous Envs as immunogens, but to date has not found a regimen to induce high levels of bnAbs. 
     Recently, a new paradigm for design of strategies for induction of broadly neutralizing antibodies was introduced, that of B cell lineage immunogen design (Nature Biotech. 30: 423, 2012) in which the induction of bnAb lineages is recreated. It was recently demonstrated the power of mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction (Nature 496: 469, 2013). From this type of work has come the hypothesis that bnAb induction will require a selection of antigens to recreate the “swarms” of sequentially evolved viruses that occur in the setting of bnAb generation in vivo in HIV infection (Nature 496: 469, 2013). 
     Induction of HIV-1 envelope (Env) broadly neutralizing antibodies (BnAbs) is a key goal of HIV-1 vaccine development. BnAbs can target conserved regions that include conformational glycans, the gp41 membrane proximal region, the V1/V2 region, glycans-associated C3/V3 on gp120, and the CD4 binding site (CD4bs) (Walker et al, Science 326:285-289 (2009), Walker et al, Nature 477:466-470 (2011), Burton et al, Science 337:183-186 (2012), Kwong and Mascola, Immunity 37:412-425 (2012), Wu et al, Science 329:856-861 (2010), Wu et al, Science 333:1593-1602 (2011), Zhou et al, Science 329:811-817 (2010), Sattentau and McMichael, F1000 Biol. Rep. 2:60 (2010), Stamatotos, Curr. Opin. Immunol. 24:316-323 (2012)). Most mature BnAbs have one or more unusual features (long heavy chain third complementarity determining regions [HCDR3s], polyreactivity for non-HIV-1 antigens, and high levels of somatic mutation) suggesting substantial barriers to their elicitation (Kwong and Mascola, Immunity 37:412-425 (2012), Haynes et al, Science 308:1906-1908 (2005), Haynes et al, Nat. Biotechnol. 30:423-433 (2012), Mouquet and Nussenzweig, Cell Mol. Life Sci. 69:1435-1445 (2012), Scheid et al, Nature 458:636-640 (2009)). In particular, CD4bs BnAbs have extremely high levels of somatic mutation suggesting complex or prolonged maturation pathways (Kwong and Mascola, Immunity 37:412-425 (2012), Wu et al, Science 329:856-861 (2010), Wu et al, Science 333:1593-1602 (2011), Zhou et al, Science 329:811-817 (2010)). Moreover, it has been difficult to find Envs that bind with high affinity to BnAb germline or unmutated common ancestors (UCAs), a trait that would be desirable for candidate immunogens for induction of BnAbs (Zhou et al, Science 329:811-817 (2010), Chen et al, AIDS Res. Human Retrovirol. 23:11 (2008), Dimitrol, MAbs 2:347-356 (2010), Ma et al, PLoS Pathog. 7:e1002200 (2001), Pancera et al, J. Virol. 84:8098-8110 (2010), Xiao et al, Biochem. Biophys. Res. Commun. 390:404-409 (2009)). Whereas it has been found that Envs bind to UCAs of BnAbs targeting gp41 membrane proximal region (Ma et al, PLoS Pathog. 7:e1002200 (2001), Alam et al, J. Virol. 85:11725-11731 (2011)), and to UCAs of some V1/V2 BnAb (Bonsignori et al, J. Virol. 85:9998-10009 (2011)), to date, heterologous Envs have not been identified that bind the UCAs of CD4bs BnAb lineages (Zhou et al, Science 329:811-817 (2010), Xiao et al, Biochem. Biophys. Res. Commun. 390:404-409 (2009), Mouquet et al, Nature 467:591-595 (2010), Scheid et al, Science 333:1633-1637 (2011), Hoot et al, PLoS Pathog. 9:e1003106 (2013)), although Envs that bind CD4bs BnAb UCAs should exist (Hoot et al, PLoS Pathog. 9:e1003106 (2013)). 
     Eighty percent of heterosexual HIV-1 infections are established by one transmitted/founder (T/F) virus (Keele et al, Proc. Natl. Acad. Sci. USA 105:7552-7557 (2008)). The initial neutralizing antibody response to this virus arises approximately 3 months after transmission and is strain-specific (Richman et al, Proc. Natl. Acad. Sci. USA 100:4144-4149 (2003), Corti et al, PLoS One 5:e8805 (2010)). This antibody response to the T/F virus drives viral escape, such that virus mutants become resistant to neutralization by autologous plasma (Richman et al, Proc. Natl. Acad. Sci. USA 100:4144-4149 (2003), Corti et al, PLoS One 5:e8805 (2010)). This antibody-virus race leads to poor or restricted specificities of neutralizing antibodies in ˜80% of patients; however in ˜20% of patients, evolved variants of the T/F virus induce antibodies with considerable neutralization breadth, e.g. BnAbs (Walker et al, Nature 477:466-470 (2011), Bonsignori et al, J. Virol. 85:9998-10009 (2011), Corti et al, PLos One 5:e8805 (2010), Gray et al, J. Virol. 85:4828-4840 (2011), Klein et al, J. Exp. Med. 209:1469-1479 (2012), Lynch et al, J. Virol. 86:7588-7595 (2012), Moore et al, Curr. Opin. HIV AIDS 4:358-363 (2009), Moore et al, J. Virol. 85:3128-3141 (2011), Tomaras et al, J. Virol. 85:11502-11519 (2011)). 
     There are a number of potential molecular routes by which antibodies to HIV-1 may evolve and, indeed, types of antibodies with different neutralizing specificities may follow different routes (Wu et al, Science 333:1593-1602 (2011), Haynes et al, Nat. Biotechnol. 30:423-433 (2012), Dimitrol, MAbs 2:347-356 (2010), Liao et al, J. Exp. Med. 208:2237-2249 (2011)). Because the initial autologous neutralizing antibody response is specific for the T/F virus (Moore et al, Curr. Opin. HIV AIDS 4:358-363 (2009)), some T/F Envs might be predisposed to binding the germline or unmutated common ancestor (UCA) of the observed BnAb in those rare patients that make BnAbs. Thus, although neutralizing breadth generally is not observed until chronic infection, a precise understanding of the interplay between virus evolution and maturing BnAb lineages in early infection may provide insight into events that ultimately lead to BnAb development. BnAbs studied to date have only been isolated from individuals who were sampled during chronic infection (Walker et al, Science 326:285-289 (2009), Burton et al, Science 337:183-186 (2012), Kwong and Mascola, Immunity 37:412-425 (2012), Wu et al, Science 329:856-861 (2010), Wu et al, Science 333:1593-1602 (2011), Zhou et al, Science 329:811-817 (2010), Bonsignori et al, J. Virol. 85:9998-10009 (2011), Corti et al, PLoS One 5:e8805 (2010), Klein et al, J. Exp. Med. 209:1469-1479 (2012)). Thus, the evolutionary trajectories of virus and antibody from the time of virus transmission through the development of broad neutralization remain unknown. 
     Vaccine strategies have been proposed that begin by targeting unmutated common ancestors (UCAs), the putative naïve B cell receptors of BnAbs, with relevant Env immunogens to trigger antibody lineages with potential ultimately to develop breadth (Wu et al, Science 333:1593-1602 (2011), Haynes et al, Nat. Biotechnol. 30:423-433 (2012), Scheid et al, Nature 458:636-640 (2009), Chen et al, AIDS Res. Human Retrovirol. 23:11 (2008), Dimitrol, MAbs 2:347-356 (2010), Ma et al, PLoS Pathog. 7:e1002200 (2001), Xiao et al, Biochem. Biophys. Res. Commun. 390:404-409 (2009), Alam et al, J. Virol. 85:11725-11731 (2011), Mouquet et al, Nature 467:591-595 (2010)). This would be followed by vaccination with Envs specifically selected to stimulate somatic mutation pathways that give rise to BnAbs. Both aspects of this strategy have proved challenging due to lack of knowledge of specific Envs capable of interacting with UCAs and early intermediate (I) antibodies of BnAbs. 
     The present invention results, at least in part, from studies that resulted in the isolation of envelopes from a patient, CH0848, who was followed from early acute HIV-1 infection phase to over five years post-transmission. During this period CH0848 developed plasma HIV-1 neutralization breadth. 
     In certain aspects the invention provides Env amino acid sequences described herein and the nucleic acids encoding these, and their use as immunogens. The envelopes to be used as immunogens in accordance with the invention can be proteins, nucleic acids, or a combination. 
     The envelopes to be used as immunogens in accordance with the invention can be expressed for example but not limited as full gp160, gp140, gp145 with transmembrane portions, gp120s, gp120 resurfaced core proteins, gp120 outer domain constructs, or other minimal gp120 constructs. 
     In accordance with the invention, immunization regimens can include sequential immunizations of Env constructs selected from those encoded by the sequences as described herein, or can involve prime and boosts of combinations of Envs, or the administration of “swarms” of such sequences. Immunogenic fragments/subunits can also be used as can encoding nucleic acid sequences. Alternatively, the transmitted founder virus Env constructs can be used as primes, followed by a boost with the transmitted founder Env and sequential additions of Envs from progressively later times after transmission in patient CH848. Further, repetitive immunization can be effected with “swarms” of CH848 Envs (for example, including various combinations of the nucleic acid sequences and encoded proteins as described here) ranging from, for example but not limited to a few envelopes, e.g. 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 Envs. 
     In one embodiment, the present invention relates to a method of activating an appropriate naïve B cell response in a subject (e.g., a human) by administering the CH0848 T/F Env or Env subunits that can include the gp160, gp145 with a transmembrane portion, gp41 and gp120, an uncleaved gp140, a cleaved gp140, a gp120, a gp120 subunit such as a resurfaced core (Wu X, Science 329:856-61 (2010)), an outerdomain, or a minimum epitope (the minimal epitope to avoid dominant Env non-neutralizing epitopes), followed by boosting with representatives of subsequently evolved CH848 Env variants either given in combination to mimic the high diversity observed in vivo during affinity maturation, or in series, using vaccine immunogens specifically selected to trigger the appropriate maturation pathway by high affinity binding to UCA and antibody intermediates (Haynes et al, Nat. Biotechnol. 30:423-433 (2012)). DNA, RNA, protein or vectored immunogens can be used alone or in combination. In one embodiment of the invention, transmitted founder virus envelope is administered to the subject (e.g., human) as the priming envelope and then one or more of the sequential envelopes disclosed herein is administered as a boost in an amount and under conditions such that BnAbs are produced in the subject (e.g., human). By way of example, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 Envs. envelopes (or subunits thereof) can be used with one prime and multiple boosts. A skilled artisan can readily determine the interval between different boosts, and the number of boosts. 
     The present invention includes the specific envelope proteins disclosed herein (e.g., those encoded by the sequences in the figures) and nucleic acids comprising nucleotide sequences encoding same. The envelope proteins (and subunits) can be expressed, for example, in 293T cells, 293F cells or CHO cells (Liao et al, Virology 353:268-82 (2006)). As indicated above, the envelope proteins can be expressed, for example, as gp120 or gp140 proteins and portions of the envelope proteins can be used as immunogens such as the resurfaced core protein design (RSC) (Wu et al, Science 329:856-861 (2010)); another possible design is an outer domain design (Lynch et al, J. Virol. 86:7588-95 (2012)). The invention includes immunogenic fragments/subunits of the envelope sequences disclosed herein, including fragments at least 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 300, 320 or more amino acids in length, as well as nucleic acids comprising nucleotide sequences encoding such fragments and vectors containing same. 
     In other embodiments, the invention provides variants of the sequences, including variants that comprise a mutation which repairs a trypsin cleavage site, thereby preventing protein clipping during Env protein production in a cell line, e.g., a CHO cell line. 
     The envelopes (immunogens) can be formulated with appropriate carriers using standard techniques to yield compositions suitable for administration. The compositions can include an adjuvant, such as, for example, alum, poly IC, MF-59 or other squalene-based adjuvant, ASO1B or other liposomal based adjuvant suitable for protein immunization. 
     As indicated above, nucleic acid sequences (e.g., DNA sequences) encoding the immunogens can also be administered to a subject (e.g., a human) under conditions such that the immunogen is expressed in vivo and BnAbs are produced. The DNA can be present as an insert in any suitable vector. Non-limiting examples of such vectors are rAdenoviral (Barouch, et al. Nature Med. 16: 319-23 (2010), recombinant mycobacterial (i.e., BCG or  M smegmatis ) (Yu et al. Clinical Vaccine Immunol. 14: 886-093 (2007); ibid 13: 1204-11 (2006), or recombinant vaccinia type of vector (Santra S. Nature Med. 16: 324-8 (2010)). 
     Immunogens of the invention, and nucleic acids (e.g., DNAs) encoding same, are suitable for use in generating an immune response (e.g., BnAbs) in a patient (e.g., a human patient) to HIV-1. The mode of administration of the immunogen, or encoding sequence, can vary with the particular immunogen, the patient and the effect sought, similarly, the dose administered. Typically, the administration route is intramuscular or subcutaneous injection (intravenous and intraperitoneal can also be used). Additionally, the formulations can be administered via the intranasal route, or intrarectally or vaginally as a suppository-like vehicle. Optimum dosing regimens can be readily determined by one skilled in the art. The immunogens (and nucleic acids encoding same) are suitable for use prophylactically, however, their administration to infected individuals may reduce viral load. 
     Recently, a method of making HIV vaccine immunogens based on their ability to bind to early members of a BnAb clonal lineage was proposed (PCT/US2012/000442). This method is termed B cell lineage immunogen design (Haynes et al. Nature Biotech. 30: 423-433 (2012)). This method is based on the use of clonal lineage antibody members as templates for design of HIV envelope proteins that bind well to lineage members. This method is based on the use of clonal lineage antibody members as templates for design of HIV envelope proteins that bind well to lineage members. This method is based on the principle that those antigens that bind best to naïve BnAb B cell receptors (the unmutated ancestors of mature BnAbs) will be the best immunogens for driving such a clonal lineage. Thus, mature antibodies are isolated, their intermediate ancestor and unmutated ancestor precursors inferred, and the clonal lineage tree reconstructed by Baysian probability statistics and maximum likelihood analysis, and then the tree antibodies are made by recombinant techniques (Haynes et al, Nature Biotech. 30:423-433 (2012)). Then, by screening Envs, or by solving antibody and Env structures and then rational design of Envs that optimally bind to clonal tree members, immunogens are designed and produced for vaccination studies (Haynes et al, Nature Biotech. 30:423-433 (2012)). 
     Regarding the choice of gp120 vs. gp160, for the genetic immunization we would normally not even consider not using gp160. However, in acute infection, gp41 non-neutralizing antibodies are dominant and overwhelm gp120 responses (Tomaras, G et al. J. Virol. 82: 12449, 2008; Liao, H X et al. JEM 208: 2237, 2011). Recently we have found that the HVTN 505 DNA prime, rAd5 vaccine trial that utilized gp140 as an immunogen, also had the dominant response of non-neutralizing gp41 antibodies. Thus, we will evaluate early on the use of gp160 vs gp120 for gp41 dominance. 
     In certain aspects the invention provides a strategy for induction of bnAbs is to select and develop immunogens designed to recreate the antigenic evolution of Envs that occur when bnAbs do develop in the context of infection. 
     That broadly neutralizing antibodies (bnAbs) occur in nearly all sera from chronically infected HIV-1 subjects suggests anyone can develop some bnAb response if exposed to immunogens via vaccination. Working back from mature bnAbs through intermediates enabled understanding their development from the unmutated ancestor, and showed that antigenic diversity preceded the development of population breadth. See Liao et al. (2013) Nature 496, 469-476. 
     The invention provides various methods to choose a subset of viral variants, including but not limited to envelopes, to investigate the role of antigenic diversity in serial samples. Neutralization and binding methods using sera, antibodies, and suitable viruses and envelopes are known in the art. In other aspects, the invention provides compositions comprising viral variants, for example but not limited to gp160 envelopes, selected based on various criteria as described herein to be used as immunogens. 
     In other aspects, the invention provides immunization strategies using the selections of immunogens to induce cross-reactive neutralizing antibodies. In certain aspects, the immunization strategies as described herein are referred to as “swarm” immunizations to reflect that multiple envelopes are used to induce immune responses. The multiple envelopes in a swarm could be combined in various immunization protocols of priming and boosting. 
     The invention provides an approach to select reagents for neutralization assays and subsequently investigate affinity maturation, autologous neutralization, and the transition to heterologous neutralization and breadth. Given the sustained coevolution of immunity and escape this antigen selection based on antibody and antigen coevolution has specific implications for selection of immunogens for vaccine design. 
     In one embodiment, 100 clones were selected that represent the selected genetic and/or antigenic diversity of the CH848 envelopes. These sets of clones represent antigenic diversity by deliberate inclusion of polymorphisms that result from immune selection by neutralizing antibodies, and had a lower clustering coefficient and greater diversity in selected sites than sets sampled randomly. These selections of clones represent various levels of antigenic diversity in the HIV-1 envelope and are based on the genetic diversity of longitudinally sampled SGA envelopes, and correlated with other factors such as antigenic/neutralization diversity, and antibody coevolution. 
     Sequence Variants/Clones 
     Described herein are nucleic and amino acids sequences of HIV-1 envelopes. In certain embodiments, the described HIV-1 envelope sequences are gp160s. In certain embodiments, the described HIV-1 envelope sequences are gp120s. Other sequence variants, for example but not limited to gp145s, gp140s, both cleaved and uncleaved, gp150s, gp41s, which are readily derived from the nucleic acid and amino acid gp160 sequences. In certain embodiments the nucleic acid sequences are codon optimized for optimal expression in a host cell, for example a mammalian cell, a rBCG cell or any other suitable expression system. 
     In certain embodiments, the envelope design in accordance with the present invention involves deletion of residues (e.g., 5-11, 5, 6, 7, 8, 9, 10, or 11 amino acids) at the N-terminus. For delta N-terminal design, amino acid residues ranging from 4 residues or even fewer to 14 residues or even more are deleted. These residues are between the maturation (signal peptide, usually ending with CX, X can be any amino acid) and “VPVXXXX . . . ”. 
     In other embodiments, the delta N-design described for CH848 T/F envelope in  FIG. 4  can be used to make delta N-designs of other CH848 envelopes (See e.g.  FIG. 5 ). In certain embodiments, the invention relates generally to an immunogen, gp160, gp120 or gp140, without an N-terminal Herpes Simplex gD tag substituted for amino acids of the N-terminus of gp120, with an HIV leader sequence (or other leader sequence), and without the original about 4 to about 25, for example 11, amino acids of the N-terminus of the envelope (e.g. gp120). See WO2013/006688, e.g. at pages 10-12, the contents of which publication is hereby incorporated by reference in its entirety. 
     The general strategy of deletion of N-terminal amino acids of envelopes results in proteins, for example gp120s, expressed in mammalian cells that are primarily monomeric, as opposed to dimeric, and, therefore, solves the production and scalability problem of commercial gp120 Env vaccine production. In other embodiments, the amino acid deletions at the N-terminus result in increased immunogenicity of the envelopes. 
     In certain embodiments, the invention provides envelope sequences, amino acid sequences and the corresponding nucleic acids, and in which the V3 loop is substituted with the following V3 loop sequence TRPNNNTRKSIRIGPGQTFY ATGDIIGNIRQAH (SEQ ID NO: 1). This substitution of the V3 loop reduced product cleavage and improves protein yield during recombinant protein production in CHO cells. 
     In certain embodiments, the CH848 envelopes will have added certain amino acids to enhance binding of various broad neutralizing antibodies. 
     In certain aspects, the invention provides composition and methods which use a selection of sequential CH848 Envs, as gp120s, gp 145s, gp150s, gp 140s cleaved and uncleaved and gp160s, as proteins, DNAs, RNAs, or any combination thereof, administered as primes and boosts to elicit immune response. Sequential CH848 Envs as proteins would be co-administered with nucleic acid vectors containing Envs to amplify antibody induction. In certain embodiments, the compositions and methods include any immunogenic HIV-1 sequences to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic and/or consensus HIV-1 genes to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic group M and/or consensus genes to give the best coverage for T cell help and cytotoxic T cell induction. In some embodiments, the mosaic genes are any suitable gene from the HIV-1 genome. In some embodiments, the mosaic genes are Env genes, Gag genes, Pol genes, Nef genes, or any combination thereof. See e.g. U.S. Pat. No. 7,951,377. In some embodiments the mosaic genes are bivalent mosaics. In some embodiments the mosaic genes are trivalent. In some embodiments, the mosaic genes are administered in a suitable vector with each immunization with Env gene inserts in a suitable vector and/or as a protein. In some embodiments, the mosaic genes, for example as bivalent mosaic Gag group M consensus genes, are administered in a suitable vector, for example but not limited to HSV2, would be administered with each immunization with Env gene inserts in a suitable vector, for example but not limited to HSV-2. 
     In certain aspects the invention provides compositions and methods of Env genetic immunization either alone or with Env proteins to recreate the swarms of evolved viruses that have led to bnAb induction. Nucleotide-based vaccines offer a flexible vector format to immunize against virtually any protein antigen. Currently, two types of genetic vaccination are available for testing—DNAs and mRNAs. 
     In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See Graham B S, Enama M E, Nason M C, Gordon I J, Peel S A, et al. (2013) DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for delivery of nucleic acids, as DNA and/or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments, DNA is formulated for delivery by a gene gun. In certain embodiments, DNA is administered by electroporation, or by needle-free injection technologies, for example but not limited to Biojector® device. In certain embodiments, the DNA is inserted in vectors. The DNA is delivered using a suitable vector for expression in mammalian cells. In certain embodiments the nucleic acids encoding the envelopes are optimized for expression. In certain embodiments DNA is optimized, e.g. codon optimized, for expression. In certain embodiments the nucleic acids are optimized for expression in vectors and/or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus based vectors, rAdenovirus (Barouch D H, et al. Nature Med. 16: 319-23, 2010), recombinant mycobacteria (i.e., rBCG or  M smegmatis ) (Yu, J S et al. Clinical Vaccine Immunol. 14: 886-093, 2007; ibid 13: 1204-11, 2006), and recombinant vaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010), for example but not limited to ALVAC, replicating (Kibler K V et al., PLoS One 6: e25674, 2011 nov 9.) and non-replicating (Perreau M et al. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MVA)), adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons, Herpes Simplex Virus vectors, and other suitable vectors. 
     In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies which contemplate using DNA or RNA, or may use complexes of nucleic acid molecules and other entities to be used in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer (amphiphilic block copolymer 704). See Cany et al., Journal of Hepatology 2011 vol. 54 j 115-121; Arnaoty et al., Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology, vol. 859, pp 293-305 (2012); Arnaoty et al. (2013) Mol Genet Genomics. 2013 August; 288(7-8):347-63. Nanocarrier technologies called Nanotaxi® for immunogenic macromolecules (DNA, RNA, Protein) delivery are under development. See e.g. InCellArt research and development technologies. 
     In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins suitable for use in immunization are known in the art. 
     The immunogenic envelopes can also be administered as a protein boost in combination with a variety of nucleic acid envelope primes (e.g., HIV-1 Envs delivered as DNA expressed in viral or bacterial vectors). 
     Dosing of proteins and nucleic acids can be readily determined by a skilled artisan. A single dose of nucleic acid can range from a few nanograms (ng) to a few micrograms (μg) or milligram of a single immunogenic nucleic acid. Recombinant protein dose can range from a few μg micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide. 
     Administration: The compositions can be formulated with appropriate carriers using known techniques to yield compositions suitable for various routes of administration. In certain embodiments the compositions are delivered via intramascular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes. 
     In certain embodiment, nucleic acids and proteins could be administered together, either in the same formulation or in different formulations, or could be administered simultaneously at the same or different immunization sites. 
     The compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to, alum, poly IC, MF-59 or other squalene-based adjuvant, ASOIB, or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, TLR agonists are used as adjuvants. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions. In certain embodiments, the methods and compositions comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In non-limiting embodiments modulation includes PD-1 blockade; T regulatory cell depletion; CD40L hyperstimulation; soluble antigen administration, wherein the soluble antigen is designed such that the soluble agent eliminates B cells targeting dominant epitopes, or a combination thereof. In certain embodiments, an immunomodulatory agent is administered in at time and in an amount sufficient for transient modulation of the subject&#39;s immune response so as to induce an immune response which comprises broad neutralizing antibodies against HIV-1 envelope. Non-limiting examples of such agents is any one of the agents described herein: e.g. chloroquine (CQ), PTP1B Inhibitor-CAS 765317-72-4-Calbiochem or MSI 1436 clodronate or any other bisphosphonate; a Foxo1 inhibitor, e.g. 344355 |Foxo1 Inhibitor, AS1842856-Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, an agent which binds to a B cell receptor for a dominant HIV-1 envelope epitope, or any combination thereof. In certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different. 
     There are various host mechanisms that control bNAbs. For example highly somatically mutated antibodies become autoreactive and/or less fit (Immunity 8: 751, 1998; PloS Comp. Biol. 6 e1000800, 2010; J. Thoret. Biol. 164:37, 1993); Polyreactive/autoreactive naïve B cell receptors (unmutated common ancestors of clonal lineages) can lead to deletion of Ab precursors (Nature 373: 252, 1995; PNAS 107: 181, 2010; J. Immunol. 187: 3785, 2011); Abs with long HCDR3 can be limited by tolerance deletion (JI 162: 6060, 1999; JCI 108: 879, 2001). BnAb knock-in mouse models are providing insights into the various mechanisms of tolerance control of MPER BnAb induction (deletion, anergy, receptor editing). Other variations of tolerance control likely will be operative in limiting BnAbs with long HCDR3s, high levels of somatic hypermutations. 2F5 and 4E10 BnAbs were induced in mature antibody knock-in mouse models with MPER peptide-liposome-TLR immunogens. Next step is immunization of germline mouse models and humans with the same immunogens. 
     Table 1 below summarizes sequences listed in  FIGS. 5, 6, 7, and 8 . 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Plasmid 
                   
                 Plasmid 
                   
               
               
                   
                 ID 
                 Env Name 
                 ID 
                 Env Name 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1. 
                 HV1300892 
                 CH848.3.d0078.30.02D11gp120 
                 HV1300815 
                 CH848.3.d0078.30.02gp140C 
               
               
                 2. 
                 HV1300893 
                 CH848.3.d0078.30.42D11gp120 
                 HV1300816 
                 CH848.3.d0078.30.42gp140C 
               
               
                 3. 
                 HV1300894 
                 CH848.3.d0107.30.12D11gp120 
                 HV1300817 
                 CH848.3.d0107.30.12gp140C 
               
               
                 4. 
                 HV1300895 
                 CH848.3.d0107.30.27D11gp120 
                 HV1300818 
                 CH848.3.d0107.30.27gp140C 
               
               
                 5. 
                 HV1300896 
                 CH848.3.d0107.30.31D11gp120 
                 HV1300819 
                 CH848.3.d0107.30.31gp140C 
               
               
                 6. 
                 HV1300897 
                 CH848.3.d0135.27.03D11gp120 
                 HV1300820 
                 CH848.3.d0135.27.03gp140C 
               
               
                 7. 
                 HV1300898 
                 CH848.3.d0135.27.06D11gp120 
                 HV1300821 
                 CH848.3.d0135.27.06gp140C 
               
               
                 8. 
                 HV1300899 
                 CH848.3.d0135.60.05D11gp120 
                 HV1300822 
                 CH848.3.d0135.60.05gp140C 
               
               
                 9. 
                 HV1300900 
                 CH848.3.d0135.60.14D11gp120 
                 HV1300823 
                 CH848.3.d0135.60.14gp140C 
               
               
                 10 
                 HV1300901 
                 CH848.3.d0135.60.19D11gp120 
                 HV1300824 
                 CH848.3.d0135.60.19gp140C 
               
               
                 11 
                 HV1300902 
                 CH848.3.d0135.60.20D11gp120 
                 HV1300825 
                 CH848.3.d0135.60.20gp140C 
               
               
                 12 
                 HV1300903 
                 CH848.3.d0135.60.32D11gp120 
                 HV1300826 
                 CH848.3.d0135.60.32gp140C 
               
               
                 13 
                 HV1300904 
                 CH848.3.d0135.60.34D11gp120 
                 HV1300827 
                 CH848.3.d0135.60.34gp140C 
               
               
                 14 
                 HV1300905 
                 CH848.3.d0194.25.17D11gp120 
                 HV1300828 
                 CH848.3.d0194.25.17gp140C 
               
               
                 15 
                 HV1300906 
                 CH848.3.d0194.25.21D11gp120 
                 HV1300829 
                 CH848.3.d0194.25.21gp140C 
               
               
                 16 
                 HV1300907 
                 CH848.3.d0194.25.24D11gp120 
                 HV1300830 
                 CH848.3.d0194.25.24gp140C 
               
               
                 17 
                 HV1300908 
                 CH848.3.d0194.25.48D11gp120 
                 HV1300831 
                 CH848.3.d0194.25.48gp140C 
               
               
                 18 
                 HV1300909 
                 CH848.3.d0274.30.02D11gp120 
                 HV1300832 
                 CH848.3.d0274.30.02gp140C 
               
               
                 19 
                 HV1300910 
                 CH848.3.d0274.30.07D11gp120 
                 HV1300833 
                 CH848.3.d0274.30.07gp140C 
               
               
                 20 
                 HV1300911 
                 CH848.3.d0274.30.09D11gp120 
                 HV1300834 
                 CH848.3.d0274.30.09gp140C 
               
               
                 21 
                 HV1300912 
                 CH848.3.d0274.30.14D11gp120 
                 HV1300835 
                 CH848.3.d0274.30.14gp140C 
               
               
                 22 
                 HV1300913 
                 CH848.3.d0358.80.03D11gp120 
                 HV1300836 
                 CH848.3.d0358.80.03gp140C 
               
               
                 23 
                 HV1300914 
                 CH848.3.d0358.80.06D11gp120 
                 HV1300837 
                 CH848.3.d0358.80.06gp140C 
               
               
                 24 
                 HV1300915 
                 CH848.3.d0358.80.17D11gp120 
                 HV1300838 
                 CH848.3.d0358.80.17gp140C 
               
               
                 25 
                 HV1300916 
                 CH848.3.d0358.80.44D11gp120 
                 HV1300839 
                 CH848.3.d0358.80.44gp140C 
               
               
                 26 
                 HV1300917 
                 CH848.3.d0445.25.04D11gp120 
                 HV1300840 
                 CH848.3.d0445.25.04gp140C 
               
               
                 27 
                 HV1300918 
                 CH848.3.d0445.25.18D11gp120 
                 HV1300841 
                 CH848.3.d0445.25.18gp140C 
               
               
                 28 
                 HV1300919 
                 CH848.3.d0445.25.26D11gp120 
                 HV1300842 
                 CH848.3.d0445.25.26gp140C 
               
               
                 29 
                 HV1300920 
                 CH848.3.d0445.30.41D11gp120 
                 HV1300843 
                 CH848.3.d0445.30.41gp140C 
               
               
                 30 
                 HV1300921 
                 CH848.3.d0445.30.42D11gp120 
                 HV1300844 
                 CH848.3.d0445.30.42gp140C 
               
               
                 31 
                 HV1300922 
                 CH848.3.d0526.25.02D11gp120 
                 HV1300845 
                 CH848.3.d0526.25.02gp140C 
               
               
                 32 
                 HV1300923 
                 CH848.3.d0526.25.09D11gp120 
                 HV1300846 
                 CH848.3.d0526.25.09gp140C 
               
               
                 33 
                 HV1300924 
                 CH848.3.d0526.25.10D11gp120 
                 HV1300847 
                 CH848.3.d0526.25.10gp140C 
               
               
                 34 
                 HV1300925 
                 CH848.3.d0526.25.11D11gp120 
                 HV1300848 
                 CH848.3.d0526.25.11gp140C 
               
               
                 35 
                 HV1300926 
                 CH848.3.d0526.25.21D11gp120 
                 HV1300849 
                 CH848.3.d0526.25.21gp140C 
               
               
                 36 
                 HV1300927 
                 CH848.3.d0526.25.32D11gp120 
                 HV1300850 
                 CH848.3.d0526.25.32gp140C 
               
               
                 37 
                 HV1300928 
                 CH848.3.d0526.25.39D11gp120 
                 HV1300851 
                 CH848.3.d0526.25.39gp140C 
               
               
                 38 
                 HV1300929 
                 CH848.3.d0611.9.02D11gp120 
                 HV1300852 
                 CH848.3.d0611.9.02gp140C 
               
               
                 39 
                 HV1300930 
                 CH848.3.d0611.20.12D11gp120 
                 HV1300853 
                 CH848.3.d0611.20.12gp140C 
               
               
                 40 
                 HV1300931 
                 CH848.3.d0611.20.14D11gp120 
                 HV1300854 
                 CH848.3.d0611.20.14gp140C 
               
               
                 41 
                 HV1300932 
                 CH848.3.d0611.20.28D11gp120 
                 HV1300855 
                 CH848.3.d0611.20.28gp140C 
               
               
                 42 
                 HV1300933 
                 CH848.3.d0700.15.06D11gp120 
                 HV1300856 
                 CH848.3.d0700.15.06gp140C 
               
               
                 43 
                 HV1300934 
                 CH848.3.d0700.15.15D11gp120 
                 HV1300857 
                 CH848.3.d0700.15.15gp140C 
               
               
                 44 
                 HV1300935 
                 CH848.3.d0700.15.29D11gp120 
                 HV1300858 
                 CH848.3.d0700.15.29gp140C 
               
               
                 45 
                 HV1300936 
                 CH848.3.d0700.27.06D11gp120 
                 HV1300859 
                 CH848.3.d0700.27.06gp140C 
               
               
                 46 
                 HV1300937 
                 CH848.3.d0794.5.27D11gp120 
                 HV1300860 
                 CH848.3.d0794.5.27gp140C 
               
               
                 47 
                 HV1300938 
                 CH848.3.d0794.5.41D11gp120 
                 HV1300861 
                 CH848.3.d0794.5.41gp140C 
               
               
                 48 
                 HV1300939 
                 CH848.3.d0836.10.36D11gp120 
                 HV1300862 
                 CH848.3.d0836.10.36gp140C 
               
               
                 49 
                 HV1300940 
                 CH848.3.d0864.7.26D11gp120 
                 HV1300863 
                 CH848.3.d0864.7.26gp140C 
               
               
                 50 
                 HV1300941 
                 CH848.3.d0864.7.39D11gp120 
                 HV1300864 
                 CH848.3.d0864.7.39gp140C 
               
               
                 51 
                 HV1300942 
                 CH848.3.d0893.10.06D11gp120 
                 HV1300865 
                 CH848.3.d0893.10.06gp140C 
               
               
                 52 
                 HV1300943 
                 CH848.3.d1120.10.13D11gp120 
                 HV1300866 
                 CH848.3.d1120.10.13gp140C 
               
               
                 53 
                 HV1300944 
                 CH848.3.d1120.10.21D11gp120 
                 HV1300867 
                 CH848.3.d1120.10.21gp140C 
               
               
                 54 
                 HV1300945 
                 CH848.3.d1120.10.24D11gp120 
                 HV1300868 
                 CH848.3.d1120.10.24gp140C 
               
               
                 55 
                 HV1300946 
                 CH848.3.d1120.10.32D11gp120 
                 HV1300869 
                 CH848.3.d1120.10.32gp140C 
               
               
                 56 
                 HV1300947 
                 CH848.3.d1120.10.41D11gp120 
                 HV1300870 
                 CH848.3.d1120.10.41gp140C 
               
               
                 57 
                 HV1300948 
                 CH848.3.d1305.10.13D11gp120 
                 HV1300871 
                 CH848.3.d1305.10.13gp140C 
               
               
                 58 
                 HV1300949 
                 CH848.3.d1305.10.21D11gp120 
                 HV1300872 
                 CH848.3.d1305.10.21gp140C 
               
               
                 59 
                 HV1300950 
                 CH848.3.d1305.10.30D11gp120 
                 HV1300873 
                 CH848.3.d1305.10.30gp140C 
               
               
                 60 
                 HV1300951 
                 CH848.3.d1305.10.35D11gp120 
                 HV1300874 
                 CH848.3.d1305.10.35gp140C 
               
               
                 61 
                 HV1300952 
                 CH848.3.d1432.5.18D11gp120 
                 HV1300875 
                 CH848.3.d1432.5.18gp140C 
               
               
                 62 
                 HV1300953 
                 CH848.3.d1432.5.27D11gp120 
                 HV1300876 
                 CH848.3.d1432.5.27gp140C 
               
               
                 63 
                 HV1300954 
                 CH848.3.d1432.5.41D11gp120 
                 HV1300877 
                 CH848.3.d1432.5.41gp140C 
               
               
                 64 
                 HV1300955 
                 CH848.3.d1432.5.50D11gp120 
                 HV1300878 
                 CH848.3.d1432.5.50gp140C 
               
               
                 65 
                 HV1300956 
                 CH848.3.d1432.5.56D11gp120 
                 HV1300879 
                 CH848.3.d1432.5.56gp140C 
               
               
                 66 
                 HV1300957 
                 CH848.3.d1621.4.12D11gp120 
                 HV1300880 
                 CH848.3.d1621.4.12gp140C 
               
               
                 67 
                 HV1300958 
                 CH848.3.d1621.4.15D11gp120 
                 HV1300881 
                 CH848.3.d1621.4.15gp140C 
               
               
                 68 
                 HV1300959 
                 CH848.3.d1621.4.25D11gp120 
                 HV1300882 
                 CH848.3.d1621.4.25gp140C 
               
               
                 69 
                 HV1300960 
                 CH848.3.d1621.4.31D11gp120 
                 HV1300883 
                 CH848.3.d1621.4.31gp140C 
               
               
                 70 
                 HV1300961 
                 CH848.3.d1621.4.44D11gp120 
                 HV1300884 
                 CH848.3.d1621.4.44gp140C 
               
               
                 71 
                 HV1300962 
                 CH848.3.d1621.4.46D11gp120 
                 HV1300885 
                 CH848.3.d1621.4.46gp140C 
               
               
                 72 
                 HV1300963 
                 CH848.3.d1635.10.35D11gp120 
                 HV1300886 
                 CH848.3.d1635.10.35gp140C 
               
               
                 73 
                 HV1300964 
                 CH848.3.d1651.7.34D11gp120 
                 HV1300887 
                 CH848.3.d1651.7.34gp140C 
               
               
                 74 
                 HV1300965 
                 CH848.3.d1651.7.50D11gp120 
                 HV1300888 
                 CH848.3.d1651.7.50gp140C 
               
               
                 75 
                 HV1300966 
                 CH848.3.d1651.10.04D11gp120 
                 HV1300889 
                 CH848.3.d1651.10.04gp140C 
               
               
                 76 
                 HV1300967 
                 CH848.3.d1677.521.D11gp120 
                 HV1300890 
                 CH848.3.d1677.521.gp140C 
               
               
                 77 
                 HV1300968 
                 CH848.3.d1720.5D11gp120 
                 HV1300891 
                 CH848.3.d1720.5gp140C 
               
               
                 78 
                 HV1301011 
                 CH0848d0526.25.26D11gp120 
                 HV1301062 
                 CH0848d0526.25.26gp140C 
               
               
                 79 
                 HV1301012 
                 CH0848d0700.15.34D11gp120 
                 HV1301063 
                 CH0848d0700.15.34gp140C 
               
               
                 80 
                 HV1301013 
                 CH0848d0780.25.05D11gp120 
                 HV1301064 
                 CH0848d0780.25.05gp140C 
               
               
                 81 
                 HV1301014 
                 CH0848d0700.15.05D11gp120 
                 HV1301065 
                 CH0848d0700.15.05gp140C 
               
               
                 82 
                 HV1301015 
                 CH0848d0794.3.03D11gp120 
                 HV1301066 
                 CH0848d0794.3.03gp140C 
               
               
                 83 
                 HV1301016 
                 CH0848d0836.10.31D11gp120 
                 HV1301067 
                 CH0848d0836.10.31gp140C 
               
               
                 84 
                 HV1301017 
                 CH0848d0808.15.27D11gp120 
                 HV1301068 
                 CH0848d0808.15.27gp140C 
               
               
                 85 
                 HV1301018 
                 CH0848d0949.10.18D11gp120 
                 HV1301069 
                 CH0848d0949.10.18gp140C 
               
               
                 86 
                 HV1301019 
                 CH0848d0808.15.25D11gp120 
                 HV1301070 
                 CH0848d0808.15.25gp140C 
               
               
                 87 
                 HV1301020 
                 CH0848d0864.3.03D11gp120 
                 HV1301071 
                 CH0848d0864.3.03gp140C 
               
               
                 88 
                 HV1301021 
                 CH0848d0893.10.05D11gp120 
                 HV1301072 
                 CH0848d0893.10.05gp140C 
               
               
                 89 
                 HV1301022 
                 CH0848d0949.10.10D11gp120 
                 HV1301073 
                 CH0848d0949.10.10gp140C 
               
               
                 90 
                 HV1301023 
                 CH0848d0949.10.17D11gp120 
                 HV1301074 
                 CH0848d0949.10.17gp140C 
               
               
                 91 
                 HV1301024 
                 CH0848d0808.15.15D11gp120 
                 HV1301075 
                 CH0848d0808.15.15gp140C 
               
               
                 92 
                 HV1301025 
                 CH0848d0780.15.22D11gp120 
                 HV1301076 
                 CH0848d0780.15.22gp140C 
               
               
                 93 
                 HV1301026 
                 CH0848d0780.15.29D11gp120 
                 HV1301077 
                 CH0848d0780.15.29gp140C 
               
               
                 94 
                 HV1301027 
                 CH0848d0808.15.43D11gp120 
                 HV1301078 
                 CH0848d0808.15.43gp140C 
               
               
                 95 
                 HV1301028 
                 CH0848d1120.10.05D11gp120 
                 HV1301079 
                 CH0848d1120.10.05gp140C 
               
               
                 96 
                 HV1301029 
                 CH0848d1432.5.06D11gp120 
                 HV1301080 
                 CH0848d1432.5.06gp140C 
               
               
                 97 
                 HV1301030 
                 CH0848d1432.5.48D11gp120 
                 HV1301081 
                 CH0848d1432.5.48gp140C 
               
               
                 98 
                 HV1301031 
                 CH0848d1432.5.35D11gp120 
                 HV1301082 
                 CH0848d1432.5.35gp140C 
               
               
                 99 
                 HV1301032 
                 CH0848d1651.10.07D11gp120 
                 HV1301083 
                 CH0848d1651.10.07gp140C 
               
               
                 10 
                 HV1301058 
                 CH0848d1432.5.26D11gp120 
                 HV1301084 
                 CH0848d1432.5.26gp140C 
               
               
                   
               
            
           
         
       
     
     The invention is further described in the non-limiting examples below. 
     EXAMPLES 
     Example 1 
     Provided herein are non-limiting examples of combinations of antigens derived from CH848 envelope sequences for a swarm immunization. The selection includes priming with a virus which binds to a UCA, for example a T/F virus or another early virus envelope. In certain embodiments the prime could include D-loop variants. 
     Non-limiting embodiments of envelopes selected for swarm vaccination are shown as the selections described below. A skilled artisan would appreciate that a vaccination protocol can include a sequential immunization starting with the “prime” envelope(s) and followed by sequential boosts, which include individual envelopes or combination of envelopes. In another vaccination protocol, the sequential immunization starts with the “prime” envelope(s) and is followed with boosts of cumulative prime and/or boost envelopes. In certain embodiments, there is some variance in the immunization regimen; in some embodiments, the selection of HIV-1 envelopes may be grouped in various combinations of primes and boosts, either as nucleic acids, proteins, or combinations thereof. In certain embodiments the immunization includes a prime administered as DNA, and MVA boosts. See Goepfert, et al. 2014; “Specificity and 6-Month Durability of Immune Responses Induced by DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles” J Infect Dis. 2014 Feb. 9. [Epub ahead of print]. 
     In a non-limiting embodiment, the immunization protocol is the following: prime with T/F, and then boost with the next 15 envelopes, then boost with the next 17 envelopes, then boost with the next 34 envelopes and then boost with the next 33 envelopes ( FIG. 1 ). 
     The contents of all documents and other information sources cited herein are incorporated by reference in their entirety.