WO2015048438A1 - Hiv-1 immunogens - Google Patents

Abstract

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).

Description

HIV-1 IMMUNOGENS

The present application claims benefit of U.S. Provisional Application No. 61/884,014 filed September 28, 2013, the entire contents of which is hereby 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 V1 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 329:811-817 (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

5 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 (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 i o 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:el002200 (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

15 UCAs of BnAbs targeting gp41 membrane proximal region (Ma et al, PLoS

Pathog. 7:el002200 (2001), Alam et al, J. Virol. 85: 1 1725-1 1731 (201 1)), and to UCAs of some VI N2 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.

20 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:el003106 (2013)), although Envs that bind CD4bs BnAb UCAs should exist (Hoot et al, PLoS Pathog. 9:e 1003106 (2013)).

Eighty percent of heterosexual HIV-1 infections are established by one

25 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 (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. 85:3128-3141

(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 Retroviral. 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 (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 HIV Envelope encoding sequences from an individual, CHI 754, who developed plasma HIV-1 neutralization breadth over a two to three year period.

SUMMARY OF THE INVENTION

In general, the present invention relates to HIV-1. More specifically, the invention relates 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). Objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF SEQUENCE LISTING

Figure 1 of the priority U.S. Application No. 61/884,014, filed September 28, 2013, incorporated herein by reference, provides Envelope gene sequences for CH1754.

The sequences of Figure 1 of the priority document are filed herewith as a part of the Sequence Listing, in IBM-PC machine format, as a file named "1579- 2033_SL.txt" which is 3,849 KB and was created on September 26, 2014. Two (2) Compact Disc-Read Only Memory (CD-ROM) or Compact Disc-Recordable (CD-R) (labeled as "Copy 1" and "Copy 2" - in compliance with 37 CFR 1.52(e) - are being filed separately by hand carry on September 26, 2014, each disc containing a copy of the file named "1579-2033_SL.txt" which is 3,849 KB and was created on September 26, 2014, the entire contents of which file and Compact Discs are incorporated herein by reference. The two Compact Discs are identical.

DETAILED DESCRIPTION OF THE INVENTION

The present invention results, at least in part, from studies that resulted in the isolation of Envelope gene sequences from an individual, CHI 754, who developed plasma HIV-1 neutralization breadth over a two to three year period. The invention relates the Env gene sequences shown in the Sequence Listing and the encoded amino acid sequences, and the use thereof as immunogens.

The envelopes to be used as immunogens in accordance with the invention can be expressed as full gpl40, gpl45 with transmembrane portions, gpl20s, gpl20 resurfaced core proteins, gpl20 outer domain constructs, or other minimal gpl20 constructs. In accordance with the invention, immunization regimens can include sequential immunizations of Env constructs selected from those encoded by the sequences of the Sequence Listing, 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 CHI 754. Further, repetitive immunization can be effected with "swarms" of CHI 754 Envs (for example, including various combinations of the nucleic acid sequences in the Sequence Listing and encoded proteins) 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 CHI 754 T/F Env or Env submits that can include the gpl45 with a transmembrane portion, gp41 and gpl20, an uncleaved gpl40, a cleaved gpl40, a gpl20, a gpl20 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 CHI 754 Env variants (e.g., see the Sequence Listing) 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, 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 encoded by the sequences in the Sequence Listing) and nucleic acids comprising nucleotide sequences encoding same (e.g., those in the Sequence Listing). 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 the Sequence Listing, 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, ASOIB 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 intrarectal ly 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.

****

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, 2013, Provisional Appln. No. 61/883,561 , filed September 27, 2013 and PCT/US2013/059515, filed September 12, 2013.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an HIV-1 envelope protein encoded by a sequence set forth in the Sequence Listing, or immunogenic subunit thereof, and a carrier.

2. The composition according to claim 1 wherein said composition comprises the gpl20 subunit of an HIV-1 envelope protein encoded by a sequence set forth in the Sequence Listing.

3. The composition according to claim 1 wherein said composition further comprises an adjuvant.

4. A construct comprising a nucleotide sequence encoding an HIV-1 envelope protein, as set forth in the Sequence Listing, or immunogenic subunit thereof, wherein said nucleotide sequence is present in a vector.

5. The construct according to claim 4 wherein said vector is a viral vector or mycobacterial vector.

6. The construct according to claim 5 wherein said vector is an adenoviral vector or a pox virus vector.

7. A composition comprising the construct according to claim 4 and a carrier.

8. A method of inducing an immune response comprising administering to a mammal in need thereof the composition according to claim 1 in an amount sufficient to effect said induction.

9. The method according to claim 8 wherein said composition is administered by injection.

10. The method according to claim 8 wherein said composition is administered intrarectally or vaginally.

1 1. A method of inducing an immune response comprising administering to a mammal in need thereof the construct according to claim 4 under conditions such that said nucleotide sequence is expressed, said HIV-1 envelope protein, or subunit thereof, is produced and said response is induced.

12. The method according to claim 8 or 1 1 wherein said mammal is a human.