The present application claims benefit of U.S. Provisional Application No. 61/884,029, filed September 28, 2013, the entire a content of which is

incorporated herein by reference.

This invention was made with government support under Grants

All 067854 and All 00645 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to HIV-1 and, in particular, to HIV-1 immunogens and to methods of using such immunogens to induce the production of broadly neutralizing HIV-1 antibodies in a subject (e.g., a human).

BACKGROUND

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 V 1/V2 region, glycans-associated C3/V3 on gpl20, and the CD4 binding site (CD4bs) (Walker et al, Science 326:285-289 (2009), Walker et al, Nature 477:466-470 (201 1), 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 (201 1), Zhou et al, Science 329:81 1 -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 : 1 593- 1602 (201 1 ), Zhou et al, Science 329:81 1 -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 329:81 1 -817 (2010), Chen et al, AIDS Res. Human Retrovirol. 23 : 1 1 (2008), Dimitrol, MAbs 2:347-356 (2010), Ma et al, PLoS Pathog. 7:el 002200 (2001 ), Pancera et al, J. Virol. 84:8098-81 10 (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:el 002200 (2001 ), Alam et al, J. Virol. 85: 1 1725-1 1731 (201 1 )), and to UCAs of some V1 V2 BnAb (Bonsignori et al, J. Virol. 85:9998-10009 (201 1 )), to date, heterologous Envs have not been identified that bind the UCAs of CD4bs BnAb lineages (Zhou et al, Science 329:81 1 -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 (201 1 ), Hoot et al, PLoS Pathog. 9:e 1003106 (2013)), although Envs that bind CD4bs BnAb UCAs should exist (Hoot et al, PLoS Pathog. 9:e l 003 106 (2013)).

Eighty percent of heterosexual HIV- 1 infections are established by one transmitted/founder (T/F) virus ( eele 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 (201 1 ), Bonsignori et al, J. Virol. 85:9998-10009 (201 1 ), Corti et al, PLos One 5:e8805

(2010) , Gray et al, J. Virol. 85:4828-4840 (201 1), 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. 85:3 128-3141

(201 1 ) , Tomaras et al, J. Virol. 85: 1 1502-1 1519 (201 1 )).

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 (201 1 ), Haynes et al, Nat. Biotechnol. 30:423-433 (2012), Dimitrol, MAbs 2:347- 356 (2010), Liao et al, J. Exp. Med. 208:2237-2249 (201 1 )). 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 (201 1 ), Zhou et al, Science 329:81 1 -817 (2010), Bonsignori et al, J. Virol , 85 :9998- 10009 (201 1 ), 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 naive 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 (201 1 ), 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: 1 1 (2008), Dimitrol, MAbs 2:347-356 (2010), Ma et al, PLoS Pathog. 7:el002200 (2001 ), Xiao et al, Biochem. Biophys. Res. Commun. 390:404-409 (2009), Alam et al, J. Virol. 85: 1 1725- 1 1731 (201 1 ), 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 CH I 03 CD4bs BnAb clonal lineage was isolated from an African patient, CH505, who was followed from acute HIV-1 infection through BnAb development. The studies show that the CHI 03 BnAb lineage is less mutated than most other CD4 binding site BnAbs, and may be first detectable by as early as 14 weeks after HIV- 1 infection. Early autologous neutralization by antibodies in this lineage triggered virus escape, but rapid and extensive Env evolution in and near the epitope region preceded the acquisition of plasma antibody neutralization breadth defined as neutralization of heterologous viruses. Analysis of the cocrystal structure of the CH I 03 Fab and a gpl20-core demonstrated a novel loop binding mode of antibody neutralization. (See International

Applications PCT/US 13/00210 and PCT US2013/059515, both filed September 12, 2013, the entire contents of both of which are incorporated herein by refererence.) The present invention results, at least in part, from studies demonstrating that a single resistance signature mutation that was in the contact residue can, by itself, confer resistance to antibodies in the CH I 03 lineage.

SUMMARY OF THE INVENTION

In general, the present invention relates to HIV- 1 . More specifically, the invention relates to HIV- 1 immunogens and compositions comprising same. The invention further relates to methods of inducing the production of broadly neutralizing HIV-1 antibodies in a subject (e.g., a human) and to compounds and compositions suitable for use in such methods.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . Sequences of escape mutants.

Figure 2. Mutations.

DETAILED DESCRIPTION

The invention relates to the Env sequences shown in Figure 1 and to nucleic acids comprising nucleotide sequences encoding same. The invention further relates to methods of using such amino acid and nucleic acids as immunogens.

The "escape mutants" of Figure 1 are Env sequences which were designed to capture virus sequences which are not recognized by the CHI 03 antibodies. The expectation is that adding these escape mutant antigen to the polyvalent vaccine described in International Applications PCT/US 13/00210 and

PCT/US2013/059515 would increase the breadth of immunogenicity and protection.

The envelopes to be used as immunogens in accordance with the invention can be expressed as full gp!40, gpl 45 with transmembrane portions, gp l 20s, gp! 20 resurfaced core proteins, gpl 20 outer domain constructs, or other minimal gp] 20 constructs.

In accordance with the invention, immunization regimens can include sequential immunizations of Env constructs, including Envs selected from those included Fig. 1 , 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 CH0505, Further, repetitive immunization can be effected with "swarms" of CH05O5 Envs (for example, including various combinations of the sequences in Fig.1 and encoding nucleic acids) ranging from, for example, 2 to 40 Envs.

In one embodiment, the present invention relates to a method of activating an appropriate naive B cell response in a subject (e.g., a human) by administering the T/F Env or Env subunits that can include the gpl 45 with a transmembrane portion, gp41 and gpl 20, an uncleaved gpl 40, a cleaved gpl 40, a gpl 20, a gpl 20 subunit such as a resurfaced core (Wu X, Science 329:856-61 (2010)), an outerdomain, or a minimum epitope expressing only contact points of broadly neutralizing antibodies (Bnabs) with Env (the minimal epitope to avoid dominant Env non-neutralizing epitopes), followed by boosting with representatives of subsequently evolved 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 and/or in International Application PCT/US 13/00210 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, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 envelopes (or subunits thereof) can be used with one prime and multiple boosts.

The present invention includes the specific envelope proteins disclosed herein (e.g., those in Fig. 1 ) and nucleic acids comprising nucleotide sequences encoding same. The envelope proteins (and subunits thereof) 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 gpl20 or gpl40 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 encoded by the sequences in Fig. 1 , 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, ASO l B 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 a vector, such as a 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 : 1204-1 1 (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. EXAMPLE 1

A large panel of neutralizing antibodies has been tested against a large global panel of pseudotyped Envelopes. Phylogenetically corrected amino acid signature patterns have been defined that are associated with either neutralization sensitivity or resistance. Signatures have been defined for each of four major classes of neutralizing antibodies: CD4bs, VI V2 glycan (PG9-like) V3 glycan (PGT121 -like) and MPER. In an experimental validation of these signatures, 5 specific resistance signatures that were found for the CHAVI CD4bs neutralizing antibody CH I 03 were tested experimentally for impact through site-directed mutagenesis. 1 of the 5 signature sites was in a contact residue for this antibody, and could independently confer resistance, The other 4 were outside the epitope region. These did not confer resistance on their own, but combinations of 2 or 3 these mutations resulted in a CH I 03 resistant Env. The MPER signature patterns have been used to inform selection of complementary versions of the peptide for a polyvalent MPER vaccine. Use will be made of the full set of

resistance/sensitivity signature patterns to attempt to improve the M group Con S vaccine, working from the Con S baseline because it has shown promise in animal studies.

The entire contents of all documents and other information sources cited herein are hereby incorporated by reference, including, Provisional Appln.

61/613,222, filed March 20, 2012, Provisional Application No. 61/700,234, filed September 12, 2012, Provisional Application No. 61/700,252, filed September 12, 2012, Provisional Application No. 61/708,466, filed October 1 , 2012, Provisional Application No. 61/764,421 , filed February 13, 2013, Provisional Application No. 61/542,469, filed October 3, 201 1 , Provisional Application No. 61/708,413, filed October 1 , 2012, Provisional Application No, 61/708,503, filed October 1 , 2012, Provisional Application No, 61/806,717, filed March 29, 2013, U.S.

Application No. 13/314,712, filed December 8, 201 1 , PCT US2012/000442, filed October 3, 2012, PCT/US2013/00210, filed September 12, 201 3, Provisional Appln. No. 61/883,561 , filed September 27, 2013 and PCT/US201 3/059515, filed September 12, 2013.