US20170281749A1

US20170281749A1 - Compositions comprising ch505 envelopes, and trimers

Info

Publication number: US20170281749A1
Application number: US15/513,539
Authority: US
Inventors: Barton F. Haynes; Hua-Xin Liao; Bette T. Korber; Peter T. HRABER
Original assignee: Los Alamos National Security LLC; Duke University
Current assignee: Los Alamos National Security LLC; Duke University
Priority date: 2014-09-28
Filing date: 2015-09-25
Publication date: 2017-10-05
Also published as: EP3197488A2; WO2016049522A2; US10232034B2; WO2016049522A3; EP3197488A4; CA2962938A1

Abstract

In certain aspects the invention provides a selection of HIV-1 envelopes suitable for use as immunogens, and methods of using these immunogens to induce neutralizing antibodies. In certain embodiments, the immunogens are designed to trimerize. In other embodiments, the immunogens comprise an immune modulating component.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to the U.S. Provisional Patent Application No. 62/056,602, entitled “Compositions Comprising CH505 Envelopes, and Trimers” filed on Sep. 28, 2014, the contents of each of which are hereby incorporated by reference in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

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

In certain aspects the invention is directed to HIV-1 envelopes which are designed as fusion molecules comprising a portion of an envelope protein and a trimerization domain so as to trimerize. In certain aspects the invention is directed towards methods of using such HIV-1 envelopes for immunization so as to induce immune response, which comprises humoral immune response. In certain embodiments, the methods of immunization comprise administering an agent which transiently modulates the immune response.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.M6, CH505.M11, CH505w020.14, CH505w030.28, CH505w078.15, CH505w053.31, CH505w030.21, CH505w078.33, CH505w053.16, CH505w100.B6 or a combination thereof, or polynucleotide encoding the same.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.T/F; CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6 or a combination thereof, or a polynucleotide encoding the same.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.M11, CH505.w004.03, CH505.w020.14, CH505.w030.28, CH505.w030.12, CH505.w020.2, CH505.w030.10, CH505.w078.15, CH505.w030.19 CH505.w030.21 or a combination thereof, or polynucleotide encoding the same.
In certain aspects the invention provides, a composition comprising any one of the polynucleotides in Table 4 or Table 5 or a combination thereof. In some embodiments the compositions comprises envelope proteins. In some embodiments, the composition comprises polynucleotides encoding the envelope proteins.
In certain embodiments, the proteins or polynucleotides comprise a trimerization domain. In certain embodiments the trimerziation domain is GCN4. In certain embodiments the trimerization domain is CD40L. In certain embodiments the trimerization domain is linked to the envelope sequence via a linker. In certain embodiments the linker is about 6 amino acids. In other embodiments the linker is about 3-20 amino acids. In certain embodiments, the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids.
In certain embodiments, the proteins or polynucleotides further comprises a CD40L sequence.
In certain aspects the invention provides a composition comprising MPER-peptide-liposome-CD40L conjugate.
In certain embodiments, the compositions of the invention further comprise an adjuvant.
In one aspect the invention provides methods of inducing an immune response in a subject comprising administering the composition of any one of the compositions in an amount sufficient to induce an immune response.
In certain embodiments, the methods comprise administering any suitable agent or transient immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In certain embodiments, the methods of the invention comprise administering chloloquine before each immunization. In certain embodiments, chloloquine is administered for about 10 days before each immunization.
In certain embodiments, the methods of the invention comprise administering anti-CD25 antibody prior or after each immunization. In certain embodiments, anti-CD25 antibody is administered for about 5 days before each immunization.
In certain embodiments, the methods of the invention comprise compositions which comprise a nucleic acid, a protein or any combination thereof. In certain embodiments, the nucleic acid encoding the envelope is operably linked to a promoter inserted in an expression vector. In certain embodiments, the protein is recombinant.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes 703010505.TF, 703010505.W53.16, 703010505.W78.33, 703010505.W100.B6 or a combination thereof, or polynucleotide encoding the same.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes 703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.21, 703010505.W20.14, 703010505.W30.28, 703010505.W30.13, 703010505.W53.31, 703010505.W78.15, 703010505.W100.B4 or a combination thereof, or polynucleotide encoding the same.
In certain aspects, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes 703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.3, 703010505.W14.8, 703010505.W14.21, 703010505.W20.7, 703010505.W20.26, 703010505.W20.9, 703010505.W20.14, 703010505.W30.28, 703010505.W30.12, 703010505.W30.19, 703010505.W30.13, 703010505.W53.19, 703010505.W53.13, 703010505.W53.31, 703010505.W78.1, 703010505.W78.15, 703010505.W100.B4 or a combination thereof, or polynucleotide encoding the same.
Also disclosed are compositions wherein each HIV-1 envelope polypeptide comprises polypeptide or polynucleotide which is a gp 41, gp 120, gp 145, gp140, gp 150 or gp 160 variant.
In certain embodiments, the HIV-1 envelopes are administered as a nucleic acid, a protein or any combination thereof. In certain embodiments, the nucleic acid encoding the envelope is operably linked to a promoter inserted in an expression vector. In certain embodiments, the protein is recombinant.
In certain embodiments, the envelopes are administered as a prime, a boost, or both. In certain embodiments, the envelopes, or any combinations thereof are administered as a multiple boosts. In certain embodiments, the compositions and method further comprise an adjuvant. In certain embodiments, the HIV-1 envelopes are provided as nucleic acid sequences, including but not limited to nucleic acids optimized for expression in the desired vector and/or host cell. In other embodiments, the HIV-1 envelopes are provided as recombinantly expressed protein.
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, for example but not limited to, as described herein. In certain embodiments, these compositions are used in immunization methods as a prime and/or boost, for example but not limited to, as described herein.
In certain embodiments, the compositions contemplate nucleic acid, as DNA and/or RNA, or protein immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and/or RNA, immunization either alone or in combination with envelope protein(s).
In certain embodiments, the nucleic acid encoding an envelope is operably linked to a promoter inserted in 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. Also provided is a nucleic acid consisting essentially of any one of the nucleic acid sequences of invention. Also provided is 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 a combination of one nucleic acid sequence encoding any one of the polypeptides of the invention. In certain embodiments, combining DNA and protein gives higher magnitude of ab responses. See Pissani F. Vaccine 32: 507-13, 2013; Jalah R et al PLoS One 9: e91550, 2014.
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 of the inventions can be a trimer. 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows designs of HIV-1 envelopes with trimerization domain, and immune modulating (e.g. CD40L) domain.

FIGS. 2A and 2B show Envelope monomer timer QC—Non-reducing conditions.

FIGS. 3A and 3B show Envelope monomer timer QC—reducing conditions.

FIG. 4 shows Envelope trimer by Blue Native PAGE.

FIG. 5 shows a summary of the data in FIGS. 6-11. FIG. 5 shows that CH505TF gp120 GCN4 Trimer binds to CH103 UCA (CD4bs) with a lower Kd (nm) compared to the CH505TF gp120 D7 Monomer.

FIG. 6 shows gp120 trimers antigenicity.

FIG. 7 shows gp120 trimers antigenicity.

FIG. 8 shows gp120 trimers antigenicity.

FIG. 9 shows gp120 trimers antigenicity.

FIG. 10 shows gp120 trimers antigenicity.

FIG. 11 shows gp120 trimers antigenicity.

FIG. 12 shows the design of CD40L-MPER656 peptide-liposome conjugate.

FIG. 13 shows antigenicity of the MPER-liposome of FIG. 12. Biolayer interferometry assay of binding of mouse anti-human CD40L mAb (A) and broadly neutralizing HIV-1 gp41 MPER mAbs 2F5 (B) and 4E10 mAbs (C) at 20 μg/ml to CD40L-MPER656 liposomes loaded onto Aminopropyl silane sensors are shown. The binding of antibodies to appropriate control liposomes were subtracted to obtain the specific binding shown in panel A-C.

FIG. 14 show that the CD40L-MPER656 peptide-liposome conjugate is functional.

FIG. 15 shows that CH505 gp120-GCN4-CD40L activates human CD40 expressing HEK cells.

FIG. 16 shows antigenicity of CH505 gp120-GCN4-CD40L. SPR binding assay essentially as described in FIG. 13.

FIG. 17 shows sequence of a selection of ten envelopes (“P10” derived from CH505).

FIG. 18A shows binding of CH103 antibodies to the autologous Envs of FIG. 17, log AUC. FIG. 18B shows the selection of 10 CH505 Envs (10 PR) based on the binding intensity for the CH103 members. Humanized mice are immunized with this selection of HIV-1 envelopes.

FIG. 19 shows neutralization IC50s of CH103 lineage mAbs against autologous CH505 Envs. Pseudoviruses are sorted by sensitivity to CH103-lineage mAbs, then geometric mean IC50. Here only 108 viruses with distinct gp120s are shown, not the full set of 135 Envs assayed.

FIG. 20 shows autologous neutralization profiles for (A) mutated TF viruses, (B) 4-Env immunogen set, (C) previously identified 10-Env immunogen set, and (D) currently identified 10-Env immunogen set. Concatenated sites listed in Table 1 are shown for each candidate immunogen

FIG. 21 CH505 (A) Env Mutations, (B) CH103 lineage MAb IC50s, and (C) Env phylogeny. Env immunogens proposed in alternative vaccination regimes are shown by colored diamonds. Unlike earlier phylogenies of these Envs, insertions and deletions here are treated as distinct characters, rather than missing data.

FIG. 22 show the sequences of four envelopes, grouped as HIV-1 Envelopes Selection C.

FIG. 23 shows the sequences of CAP 206 envelopes from 6 months.

FIG. 24 shows the sequence of ten early envelopes, grouped as HIV-1 Envelopes Selection E.

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).
A critical question is why the CH505 immunogens are better than other immunogens. This rationale comes from three recent observations. First, a series of immunizations of single putatively “optimized” or “native” trimers when used as an immunogen have not induced bnAbs as single immunogens. Second, in all the chronically infected individuals who do develop bnAbs, they develop them in plasma after ˜2 years. When these individuals have been studied at the time soon after transmission, they do not make bnAbs immediately.
Two other considerations are important. The first is that for the CH103 bnAb CD4 binding site lineage, the VH4-59 and Vλ3-1 genes are common as are the VDJ, VJ recombinations of the lineage (Liao, Nature 496: 469, 2013). In addition, the bnAb sites are so unusual, we are finding that the same VH and VL usage is recurring in multiple individuals. Thus, we can expect the CH505 Envs to induce CD4 binding site antibodies in many different individuals.
Finally, one needs to make a choice regarding gp120 vs. gp160 for the genetic immunization 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, the use of gp160 vs gp120 for gp41 dominance needs to be evaluated.
In certain aspects, the invention provides a strategy for induction of bnAbs, which involves selecting and developing immunogens designed to recreate the antigenic evolution of Envs that occur when bnAbs 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. In this study, an individual “CH505” was followed from HIV-1 transmission to development of broadly neutralizing antibodies. This individual developed antibodies targeted to CD4 binding site on gp120. In this individual the virus was sequenced over time, and broadly neutralizing antibody clonal lineage (“CH103”) was isolated by antigen-specific B cell sorts, memory B cell culture, and amplified by VH/VL next generation pyrosequencing. See Liao et al. (2013) Nature 496, 469-476.
Further analysis of envelopes and antibodies from the CH505 individual indicated that a non-CH103 Lineage participates in driving CH103-BnAb induction. For example V1 loop, V5 loop and CD4 binding site loop mutations escape from CH103 and are driven by CH103 lineage. Loop D mutations enhanced neutralization by CH103 lineage and are driven by another lineage. Transmitted/founder Env, or another early envelope for example W004.03, and/or W004.26, triggers naïve B cell with CH103 Unmutated Common Ancestor (UCA) which develop in to intermediate antibodies. Transmitted/founder Env, or another early envelope for example W004.03, and/or W004.26, also triggers non-CH103 autologous neutralizing Abs that drive loop D mutations in Env that have enhanced binding to intermediate and mature CH103 antibodies and drive remainder of the lineage.
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. In other aspects, the invention provides compositions comprising viral variants, for example but not limited to 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.

Sequences/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 sequences, for example but not limited to gp140s, both cleaved and uncleaved, gp140 Envs with the deletion of the cleavage (C) site, fusion (F) and immunodominant (I) region in gp41—named as gp140ΔCFI, gp140 Envs with the deletion of only the cleavage (C) site and fusion (F) domain—named as gp140ΔCF, gp140 Envs with the deletion of only the cleavage (C)—named gp140ΔC (See e.g. Liao et al. Virology 2006, 353, 268-282), gp145s, 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 case of CH505 T/F Env as an example, 8 amino acids (italicized and underlined in the below sequence) were deleted: MRVMGIQRNYPQWWIWSMLGFWMLMICNGMWVTVYYGVPVWKEAKTTLFCASD AKAYEKEVHNVWATHACVPTDPNPQE . . . (rest of envelope sequence is indicated as “ . . . ”). CH505 Envelopes with delta N-terminal design are referred to as D8 or ΔN8 or deltaN8. In other embodiments, the delta N-design described for CH505 T/F envelope can be used to make delta N-designs of other CH505 envelopes. 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 CH505 envelopes will have added certain amino acids to enhance binding of various broad neutralizing antibodies. Such modifications could include but not limited to, mutations at W680G or modification of glycan sites for enhanced neutralization.
In certain aspects, the invention provides composition and methods which use a selection of sequential CH505 Envs, as gp120s, 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 CH505 Envs as proteins would be co-administered with nucleic acid vectors containing Envs to amplify antibody induction.
In certain embodiments the invention provides immunogens and compositions which include immunogens as trimers. In certain embodiments, the immunogens include a trimerization domain which is not derived from the HIV-1 envelope. In certain embodiments, the trimerization domain is GCN4 (See FIGS. 1 and 2B). In other embodiments the trimerization domain is CD40L. In other embodiments, the immunogens include CD40L domain (See FIGS. 1 and 2B).
HIV-1 gp120 Trimer Vaccine Immunogens (FIG. 1):
HIV-1 Env gp120 GCN4 Trimer
HIV-1 Env gp120 GCN4 trimer is designed to be expressed as soluble recombinant trimeric HIV-1 gp120 protein. HIV-1 Env gp120 is mutated from residue R to E at the cleavage site of HIV-1 gp120 at the residue positions R503 and R511 (or any mutations at this region) to destroyed the cleavage site, a 6-residue linker (GSGSGS) (SEQ. ID. NO. 2) (the linker can be variations of 3-20 residues in length) is added to the C-terminal end of HIV-1 gp120 followed by addition of 33 amino acid residues of GCN4 sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ ID NO. 3).
HIV-1 Env gp120 GCN4 CD40L Trimer:
In certain embodiments the trimer design includes an immune co-stimulator. HIV-1 Env gp120 GCN4 CD40L trimer is designed to be expressed as soluble recombinant trimeric HIV-1 gp120 protein co-expressed with functional CD40L as immune co-stimulator. HIV-1 Env gp120 is mutated from residue R to E at the cleavage site of HIV-1 gp120 at the residue positions R503 and R511 (or any mutations at this region) to destroyed the cleavage site, a 6-residue linker (GSGSGS) (SEQ. ID NO. 2) (the linker can be variations of 3-20 residues in length) is added to the C-terminal end of HIV-1 gp120, 33 amino acid residues of GCN4 sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ ID NO 4) is added to the C terminal end of the 6-residue linker, then a 11-residue liner (GGSGGSGGSGG) (SEQ ID NO. 5) (the linker can be variations of 3-20 residues in length) is added to the C terminal end of the GCN4 domain, followed by addition of the sequence of the functional extracellular domain of the human CD40 ligand (L) E113-L261.
HIV-1 Env gp120 GCN4 CD40L Trimer with His Tag:
HIV-1 Env gp120 GCN4 CD40L trimer with His tag is designed to be expressed as soluble recombinant trimeric HIV-1 gp120 protein co-expressed with functional CD40L as immune co-stimulator. HIV-1 Env gp120 is mutated from residue R to E at the cleavage site of HIV-1 gp120 at the residue positions R503 and R511 (or any mutations at this region) to destroyed the cleavage site, a 6-residue linker (GSGSGS) (SEQ ID NO 2)(the linker can be variations of 3-20 residues in length) is added to the C-terminal end of HIV-1 gp120, 33 amino acid residues of GCN4 sequence (RMKQIEDKIEEILSKIYHIENEIARIKKLIGER) (SEQ ID NO 6) is added to the C terminal end of the 6-residue linker, a 11-residue liner (GGSGGSGGSGG) (SEQ ID NO 7) (the linker can be variations of 3-20 residues in length) is added to the C terminal end of the GCN4 domain, then the sequence of the functional extracellular domain of the human CD40 ligand (L) E113-L261 is then added followed by addition of 10 histine residues as tag (the His tag can be more or less of 10 residues). His-tag is added to anchor the HIV-1 gp120GCN4 CD40L into liposome through nickel.
In non-limiting embodiments the liposome comprises cholesterol, viral membrane lipid, anionic lipid, POPC (1-Palmitoyl-2-Oleoyl-sn-Giycero-3-Phosphocholine), POPE (1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS), DMPA (1,2-Dimyristoyl-sn-Glycero-3-Phosphate), DPPC, DPPG, Sphingomyelin, or any combination thereof. In certain embodiments the liposome comprises cholesterol. In certain embodiments the liposome comprises POPC. In certain embodiments the liposome comprises phosphatidylserine. In certain embodiments, the liposome comprises phosphatidic acid. In certain embodiments, the liposome comprises cardiolipin. In certain embodiments the liposome comprises cholesterol, POPC, POPE, and DMPA. The specific composition and ratio of the lipids in the liposome can be determined experimentally, so long as the liposome composition retains the antigenic and/or immunogenic properties of the HIV envelope. Non-limiting examples of methods to determine these properties are described herein.
Using the instant disclosure of envelope timers, any HIV-1 envelope can be designed as a trimer. In certain embodiments the HIV-1 envelope is any one of the envelopes or selection of envelopes in Application WO2014042669 (PCT/US PCT/US2013/000210), U.S. Application Ser. No. 61/955,402 (“Swarm Immunization with Envelopes form CH505” Examples 2-4, FIGS. 14-19); US Application Ser. Nos. 61/972,531 and 62/027,427 (Examples 2-3, FIGS. 18-24) the contents of which applications are herein incorporated by reference in their entirety.
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 a 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 (Santa 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, pp293-305 (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 www.incellart.com/en/research-and-development/technologies.html.
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 GO 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.
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, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen [Leroux-Roels et al., IABS Conference, Apr. 2013, 9]. In certain embodiments, TLR agonists are used as adjuvants. In some embodiments, the TLR agonist is a TLR4 agonist, such as but not limited to GLA/SE. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions. In some embodiments the adjuvant is TLR7 or a TLR7/8 agonist, or a TLR-9 agonist, or a combination thereof. See PCT/US2013/029164.
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.
In certain embodiments the immunogens and compositions of the invention comprise immunostimulatory components. In a non-limiting embodiment, the immunogen comprises a CD40L.
In certain embodiments, the compositions and methods 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'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.

EXAMPLES

Example 1: GCN4 Envelope Trimers and CD40L Containing Immunogens Bind HIV-1 Envelope Antibodies and are Functionally Active

Provided is one example of the design and formulation of liposomes that present immune-modulating CD40 ligand (CD40L) and HIV-1 gp41 neutralizing antigen. CD40L, the ligand for CD40 expressed on B-cell surface is anchored on the liposomes that had HIV-1 gp41 MPER peptide immunogen conjugated in them. Two broadly neutralizing gp41 membrane proximal external region (MPER) antibodies (2F5, 4E10) bound strongly to CD40L conjugated MPER peptide liposomes. This construct has important application as an experimental AIDS vaccine in providing immune-modulating effect to stimulate proliferation of B-cells capable of producing neutralizing antibodies targeting HIV-1 gp41 MPER region.
CD40L-gp41 MPER peptide-liposome conjugates: Recombinant CD40L with an N-terminal Histidine Tag

	(SEQ ID NO 8)
	(MGSSHHHHHH SSGLVPRGSH MQKGDQNPQI

	AAHVISEASS KTTSVLQWAE KGYYTMSNNL VTLENGKQLT

	VKRQGLYYIY AQVTFCSNRE ASSQAPFIAS LCLKSPGRFE

	RILLRAANTH SSAKPCGQQS IHLGGVFELQ PGASVFVNVT

	DPSQVSHGTG FTSFGLLKL)

was anchored to MPER peptide liposomes via His-Ni-NTA chelation by mixing CD40L with MPER656-Ni-NTA liposomes at 1:50 CD40L and Ni-NTA molar ratio (Figure-12).

The construction of MPER peptide Ni-NTA liposomes utilized the method of co-solubilization of MPER peptide having a membrane anchoring amino acid sequence and synthetic lipids 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC), 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE), 1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA), Cholesterol and 1,2-dioleoyl-sn-Glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA(Ni) at mole fractions 0.216, 35.00, 25.00, 20.00, 1.33 and 10 respectively. Appropriate amount of MPER peptide dissolved in chloroform-methanol mixture (7:3 v/v), appropriate amounts of chloroform stocks of phospholipids were dried in a stream of nitrogen followed by overnight vacuum drying. Liposomes were made from the dried peptide-lipid film in phosphate buffered saline (pH 7.4) using extrusion technology.
Biolayer interferometry (BLI) assay showed the binding of anti-human CD40L antibody to CD40L-MPER656 liposomes and confirmed the correct presentation of CD40 L on liposome surface (FIG. 13A). The broadly neutralizing HIV-1 gp41 MPER antibodies 2F5 and 4E10 bound strongly to CD40L-MPER656 liposomes (FIGS. 13B-C) and demonstrated that the CD40L co-display did not impede the presentation of the epitopes of 2F5 and 4E10 mAbs.
FIGS. 14 and 15 show that CD40L containing immunogens activate human CD40 expressing HEK cells.

Example 2—Combination of Antigens from CH505 Envelope Sequences for Immunization

Provided herein are non-limiting examples of combinations of antigens derived from CH505 envelope sequences for a swarm immunization. The selection includes priming with a virus which binds to the UCA, for example a T/F virus or another early (e.g. but not limited to week 004.3, or 004.26) virus envelope. In certain embodiments the prime could include D-loop variants. In certain embodiments the boost 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, the prime does not include T/F sequence (W000.TF). In certain embodiments, the prime includes w004.03 envelope. In certain embodiments, the prime includes w004.26 envelope. In certain embodiments, the immunization methods do not include immunization with HIV-1 envelope T/F. In other embodiments for example the T/F envelope may not be included when w004.03 or w004.26 envelope is included. 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].
HIV-1 Envelope selection A (ten envelopes: sensitive envelopes): 703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.21, 703010505.W20.14, 703010505.W30.28, 703010505.W30.13, 703010505.W53.31, 703010505.W78.15, 703010505.W100.B4, optionally in certain embodiments designed as trimers. See U.S. Provisional Application No. 62/027,427 incorporated by reference.
HIV-1 Envelope selection B (twenty envelopes: sensitive envelopes): 703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.3, 703010505.W14.8, 703010505.W14.21, 703010505.W20.7, 703010505.W20.26, 703010505.W20.9, 703010505.W20.14, 703010505.W30.28, 703010505.W30.12, 703010505.W30.19, 703010505.W30.13, 703010505.W53.19, 703010505.W53.13, 703010505.W53.31, 703010505.W78.1, 703010505.W78.15, 703010505.W100.B4, optionally in certain embodiments designed as trimers. See U.S. Provisional Application No. 62/027,427 incorporated by reference.
HIV-1 Envelope selection C (four envelopes): 703010505.TF, 703010505.W53.16, 703010505.W78. 33, 703010505.W100.B6, optionally in certain embodiments designed as trimers. See WO2014042669.
HIV-1 Envelope selection D (ten production envelopes): CH505.M6; CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6, optionally in certain embodiments designed as trimers. See FIG. 17.
HIV-1 Envelopes selection E (ten early envelopes): optionally in certain embodiments designed as trimers. CH505.M11; CH505.w004.03; CH505.w020.14; CH505.w030.28; CH505.w030.12; CH505.w020.2; CH505.w030.10; CH505.w078.15; CH505.w030.19; CH505.w030.21, optionally in certain embodiments designed as trimers. See FIG. 24.
HIV-1 Envelope selection F (ten production envelopes (10PR)): CH505.T/F; CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6, optionally in certain embodiments designed as trimers. See FIG. 18B.

Example 3: Examples of Immunization Protocols in Subjects with Swarms of HIV-1 Envelopes

Immunization protocols contemplated by the invention include envelope sequences as described herein including but not limited to nucleic acids and/or amino acid sequences of gp160s, gp150s, cleaved and uncleaved gp140s, gp120s, gp41s, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. A skilled artisan can readily modify the gp160 and gp120 sequences described herein to obtain these envelope variants. The swarm immunization protocols can be administered in any subject, for example monkeys, mice, guinea pigs, or human subjects. The swarm immunization protocols include additive and/or sequential immunization with the selections of HIV envelopes.
In non-limiting embodiments, the immunization includes a nucleic acid is administered as DNA, for example in a modified vaccinia vector (MVA). In non-limiting embodiments, the nucleic acids encode gp160 envelopes. In other embodiments, the nucleic acids encode gp120 envelopes. In other embodiments, the boost comprises a recombinant gp120 envelope. The vaccination protocols include envelopes formulated in a suitable carrier and/or adjuvant, for example but not limited to alum. In certain embodiments the immunizations include a prime, as a nucleic acid or a recombinant protein, followed by a boost, as a nucleic acid or a recombinant protein. A skilled artisan can readily determine the number of boosts and intervals between boosts.
In non-limiting embodiments, the prime includes a 703010505.TF envelope and a loop D variant as described herein. In non-limiting embodiments, the prime includes a 703010505.TF envelope and/or 703010505.W4.03, 703010505.W4.26 envelope, and a loop D variant as described herein. In certain embodiments, the loop D variant is M6. In certain embodiments, the loop D variant is M5. In certain embodiments, the loop D variant is M10. In certain embodiments, the loop D variant is M19. In certain embodiments, the loop D variant is M11. In certain embodiments, the loop D variant is M20. In certain embodiments, the loop D variant is M21. In certain embodiments, the loop D variant is M9. In certain embodiments, the loop D variant is M8. In certain embodiments, the loop D variant is M7.
Table 1 shows a non-limiting example of a sequential immunization protocol using a swarm of HIV1 envelopes (703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.21, 703010505.W20.14, 703010505.W30.28, 703010505.W30.13, 703010505.W53.31, 703010505.W78.15, 703010505.W100.B4, optionally in certain embodiments designed as trimers. In a non-limiting embodiment, a suggested grouping for prime and boost is to begin with the CH505 TF+W4.03, then boost with a mixture of w4.26+14.21+20.14, then boost with a mixture of w30.28+30.13+53.31, then boost with a mixture of w78.15+100.B4.


Envelope	Prime	Boost(s)	Boost(s)	Boost(s)

CH505 TF +	CH505 TF +
W4.03	W4.03 as a
	nucleic
	acid e.g.
	DNA/MVA
	vector and/or
	protein
w4.26 +		w4.26 +
14.21 +		14.21 +
20.14		20.14 as a
		nucleic
		acid e.g.
		DNA/MVA
		vector and/or
		protein
w30.28 +			w30.28 +
30.13 +			30.13 +
53.31			53.31 as
			a nucleic
			acid e.g.
			DNA/MVA
			vector and/or
			protein
w78.15 +				w78.15 +
100.B4				100.B4 as a
				nucleic
				acid e.g.
				DNA/MVA
				vector and/or
				protein

A skilled artisan can readily determine the number and interval between boosts.
Table 2 shows a non-limiting example of a sequential immunization protocol using a swarm of HIV1 envelopes optionally in certain embodiments designed as trimers.


Envelope	Prime	Boost(s)

703010505.TF,	703010505.TF	703010505.TF,
703010505.W4.03,	(optionally	703010505.W4.03,
703010505.W4.26,	703010505.W4.03,	703010505.W4.26,
703010505.W14.21,	703010505.W4.26)	703010505.W14.21,
703010505.W20.14,	as a nucleic acid	703010505.W20.14,
703010505.W30.28,	e.g. DNA/MVA	703010505.W30.28,
703010505.W30.13,	vector and/or	703010505.W30.13,
703010505.W53.31,	protein	703010505.W53.31,
703010505.W78.15,		703010505.W78.15,
703010505.W100.B4.		703010505.W100.B4
		as a nucleic acid
		e.g. DNA/MVA
		vector and/or
		protein

A skilled artisan can readily determine the number and interval between boosts.
For a 20mer immunization regimen (envelopes (703010505.TF, 703010505.W4.03, 703010505.W4.26, 703010505.W14.3, 703010505.W14.8, 703010505.W14.21, 703010505.W20.7, 703010505.W20.26, 703010505.W20.9, 703010505.W20.14, 703010505.W30.28, 703010505.W30.12, 703010505.W30.19, 703010505.W30.13, 703010505.W53.19, 703010505.W53.13, 703010505.W53.31, 703010505.W78.1, 703010505.W78.15, 703010505.W100.B4), in a non-limiting embodiment, one can prime with CH505 TF+W4.03, then boost with a mixture of w4.26+14.21+20.14+14.3+14.8+20.7, then boost with a mixture of w 20.26+20.9+30.12+w30.28+30.13+53.31, then boost with a mixture of w78.15+100.B4+30.19+53.19+53.13+78.1. Other combinations of envelopes are contemplated for boosts.
Table 3 shows a non-limiting example of a sequential immunization protocol using a swarm of HIV1 envelopes optionally in certain embodiments designed as trimers


Envelope	Prime	Boost(s)

703010505.TF,	703010505.TF,	703010505.TF,
703010505.W4.03,	(optionally	703010505.W4.03,
703010505.W4.26,	703010505.W4.03,	703010505.W4.26,
703010505.W14.3,	703010505.W4.26,	703010505.W14.3,
703010505.W14.8,	703010505.W14.3,	703010505.W14.8,
703010505.W14.21,	703010505.W14.8,	703010505.W14.21,
703010505.W20.7,	703010505.W14.21),	703010505.W20.7,
703010505.W20.26,	as a nucleic acid	703010505.W20.26,
703010505.W20.9,	e.g. DNA/MVA	703010505.W20.9,
703010505.W20.14,	vector and/or	703010505.W20.14,
703010505.W30.28,	protein	703010505.W30.28,
703010505.W30.12,		703010505.W30.12,
703010505.W30.19,		703010505.W30.19,
703010505.W30.13,		703010505.W30.13,
703010505.W53.19,		703010505.W53.19,
703010505.W53.13,		703010505.W53.13,
703010505.W53.31,		703010505.W53.31,
703010505.W78.1,		703010505.W78.1,
703010505.W78.15,		703010505.W78.15,
703010505.W100.B4.		703010505.W100.B4.
		as a nucleic acid
		e.g. DNA/MVA
		vector and/or
		protein

A skilled artisan can readily determine the number and interval between boosts.
Table 4 shows a non-limiting example of a sequential immunization protocol using a swarm of HIV 1 envelopes optionally in certain embodiments designed as trimers.


Envelope	Prime	Boost(s)	Boost(s)	Boost(s)

CH505.M6	CH505.M6
CH505.M11	CH505.M11
	as a nucleic
	acid e.g.
	DNA/MVA
	vector and/or
	protein
CH505w020.14		CH505w020.14
CH505w030.28		CH505w030.28
		as a nucleic
		acid e.g.
		DNA/MVA
		vector and/or
		protein
CH505w078.15			CH505w078.15
CH505w053.31			CH505w053.31
CH505w030.21			CH505w030.21as
			a nucleic
			acid e.g.
			DNA/MVA
			vector and/or
			protein
CH505w078.33				CH505w078.33
CH505w053.16				CH505w053.16
CH505w100.B6				CH505w100.B6
				as a nucleic
				acid e.g.
				DNA/MVA
				vector and/or
				protein

A skilled artisan can readily determine the number and interval between boosts.
Table 5 shows a non-limiting example of a sequential immunization protocol using a swarm of HIV 1 envelopes optionally in certain embodiments designed as trimers.


Envelope	Prime	Boost(s)	Boost(s)	Boost(s)

CH505.T/F	CH505.T/F
CH505.M11	CH505.M11
	as a nucleic
	acid e.g.
	DNA/MVA
	vector and/or
	protein
CH505w020.14		CH505w020.14
CH505w030.28		CH505w030.28
		as a nucleic
		acid e.g.
		DNA/MVA
		vector and/or
		protein
CH505w078.15			CH505w078.15
CH505w053.31			CH505w053.31
CH505w030.21			CH505w030.21as
			a nucleic
			acid e.g.
			DNA/MVA
			vector and/or
			protein
CH505w078.33				CH505w078.33
CH505w053.16				CH505w051.36
CH505w100.B6				CH505w100.B6
				as a nucleic
				acid e.g.
				DNA/MVA
				vector and/or
				protein

A skilled artisan can readily determine the number and interval between boosts.

Example 4: Selection of Ten Early Envelopes

Provided is the approach to selecting a 10-immunogen set from CH505 (See FIG. 24). Here we choose 10 low-diversity variants from the subject early on, rather than down-selecting from a short list of 18, (which you are already making) to represent diversity that appeared through week 160, and includes samples after escape from the mature CH103 mAb.
Without being bound by theory, the hypothesis is that affinity maturation in the presence of antigenic diversity helps select for breadth, allowing it to evolve gradually from a population of Envs selected by clonal autologous neutralization response. But here we would test whether modest variation in the antigen might better stimulate responses that allow the clonal lineage to interact and adapt, while the full range of variation might introduce too much diversity for the developing lineage. For example, a set of Envs with 1 or 2 substitutions in an epitope might reduce affinity, but still allow binding, and the evolving B cell population would be able to adapt. Such variants might allow more “generalists” to evolve. Env variants fully escaped from early lineage clones might be immunologically silent, and less able to draw increased breadth from the B cell clones.
This is essentially like trying a serial version of the swarm vaccine of 100, where we plan on starting with the low-diversity forms, and increase diversity as we vaccinate, but by making these 10 we could try other delivery strategies.
We selected a set of 10 gp120s for use as candidate immunogens. The focus here is on Env diversity at week 30, which coincides with an expansion in heterologous neutralization seen also by antigenic cartography. Unlike the TF and earlier forms, all week 30 sequences contain the V3 glycan shift from 334 to 332.
We identified Env sites to use as criteria for Env selection. The sites were determined by TF loss, neutralization signatures, and contact with the CD4bs and CH103 bnAb (Table 6): (a) At least 80% TF loss through week 160 yielded 36 sites, as described previously. (b) Neutralization signatures for single or PNG sites with q<0.1 for tree-corrected signatures of IC50s below 20 μg/ml, as described previously. (c) The list of contact sites was expanded by one amino acid up- and downstream of each known contact, to include a slightly larger neighborhood of contact sites. These 66 HXB2 sites grew to 71 sites when mapped onto the CH505 Env alignment. When reviewed for polymorphisms, 28 of these sites vary in CH505 over the sampling period.
CTL responses were mapped and found one ELISpot positive peptide on the C-terminus of the V4 loop, sites 409-418, EGSDTITLPC in HXB2, NSTRTITIHC in CH505. CTL epitope variants are identified among selected sites in Table 5.
Neutralization sensitivity of autologous Envs to mAbs in the CH103 lineage further informs selection of 10 Envs (FIG. 19). Comparing selected Envs with concatenated sites (FIG. 20) allows selection for incremental progression of mAb sensitivities (FIG. 20D). An abrupt transition between neutralization sensitivity to IA7 and IA3 limits available Envs from week 30 (FIG. 19), perhaps because of the mAb discontinuity induced by a shift in light-chain usage from UCA to IA2 light chain associations with IA4 and IA3 heavy chains, respectively (i.e. IA4 mAb is 14 VH and UCA VL; IA3 mAb is VH 13 with VL 12).
CH505 Env diversity and neutralization to the CH103 lineage mAbs, together with the distributions of proposed sets of 4, 10 (new and in preparation), and 100 antigens are all compared by established methods in FIG. 21.

TABLE 6

Alignment columns in Env “hot-spot” concatamer summaries.

	Col	HXB2	AA	CH505	Feature

a: 36 sites with TF loss >80%

1	279	D	N	Loop D
2	281	A	V	Loop D
3	332	O	N	PGT121
4	334	S	O	2G12
5	144+	—	—	V1
6	144+	—	—	V1
7	144+	—	—	V1
8	413	T	T	V4/CTL
9	465	S	—	V5
10	464	E	—	V5
11	417	P	H	V4/CTL
12	330	H	Y	V3
13	300	N	N	V3
14	234	O	T	8ANC195
15	302	N	K	V3
16	756	I	V	gp41
17	463+	—	—	V5
18	398	S	O	V4
19	133	D	O	V1
20	460	N	K	V5
21	347	S	K
22	275	V	E	Loop D
23	151	K	I	V1
24	356	O	H
25	471	G	G	beta24
26	147	M	O	V1
27	640	S	E	gp41
28	462	N	N	V5
29	145	G	A	V1
30	130	K	O
31	132	T	T	V1
32	620	E	G	gp41
33	4	K	M	SignalPep
34	325	N	D	V3
35	185	D	D	V2
36	412	D	R	V4/CTL

b: 28 signature sites, q < 0.1

1	130	K	O
2	132	T	T	V1
3	133	D	O	V1
4	135	K	T	V1
5	137	D	—	V1
6	146	R	S	V1
7	148	I	S	V1
8	147	M	O	V1
9	149	M	S	V1
10	151	K	I	V1
11	160	O	O	PG9
12	200	V	V
13	234	O	T	8ANC195
14	328	Q	E	V3
15	332	O	N	PGT121
16	334	S	O	2G12
17	336	A	S
18	347	S	K
19	356	O	H
20	358	T	O
21	360	I	T
22	416	L	I	V4/CTL
23	460	N	K	V5
24	461	S	O	V5
25	463	O	T	V5
26	743	D	O	Kennedy
27	745	S	S	Epitope
28	831	E	E	LLP-1

c: 28 varying contacts

1	127	V	V	CD4
2	128	S	T	CD4
3	255	V	V
4	278	T	T
5	279	D	N	Loop D
6	280	N	N	Loop D
7	281	A	V	Loop D
8	282	K	K	Loop D
9	283	T	T	Loop D
10	363	Q	P
11	365	S	S
12	367	G	G
13	369	P	L	CD4
14	371	I	I	CD4
15	372	V	T
16	424	I	I
17	433	A	A
18	460	N	K	V5
19	461	S	O	V5
20	462	N	N	V5
21	463	N	T	V5
22	463+	—	—	V5
23	463+	—	—	V5
24	463+	—	—	V5
25	463+	—	—	V5
26	463+	—	—	V5
27	464+	E	—	V5
28	471	G	G	Beta24

Example 5: Non-Human Primate Studies

NHP 79: CH505T/F gp120 envelope in GLA/SE. NHP 85: CH505T/F gp140 envelope in GLA/SE. This compares gp140 with gp120 induced antibodies.
NHP study of CH505T/F gp120 with GCN4 CH505 T/F in GLA/SE.
NHP study of CH505T/F gp120 with GCN4 CD40L CH505 T/F in GLA/SE.
NHP study of CH505T/F gp120 with GCN4 CD40L CH505 T/F in ALUM.
NHP study of CH505 T/F gp120 with GCN4 CD40L CH505 T/F=-HIS tag with liposomes in ALUM.
NHP study of M6 then rest of production 10 (Table 4) gp120 in sequence gp120 GNC4 CD40L CH505 trimers with ALUM or GLA/SE (depends on antigenicity).
NHP study of M6 then rest of production 10 (Table 4) gp120 in sequence gp120 GNC4 CD40L CH505 trimers in ALUM or GLA/SE (depends on antigenicity), with a dose of chloloquine orally each day 10 days before each immunization and then a dose of CD25 Ab 5 days after each immunization. See U.S. Application Ser. No. 62/056,583 (“Tolerance” filed concurrently), which contents is herein incorporated by reference in its entirety.
The contents of all documents and other information sources cited herein are herein incorporated by reference in their entirety.
Provided below are examples of sequences and HIV-1 envelopes disclosed in this application.
HIV-1 Envelope Selection D: Ten Production Envelopes

	TABLE 4

	FIG. 17

CH505.M6	CH505.M6D8gp120 (aa SEQ ID NO 9; nt SEQ ID
	NO: 10), CH505.M6gp145 (nt SEQ ID NO 11),
	CH505.M6 gp160 (aa SEQ ID NO: 12; nt SEQ ID
	NO 13)
CH505.M11	CH505.M11D8gp120 (aa SEQ ID NO 14; nt SEQ ID
	NO 15), CH505.M11gp145 (nt SEQ ID NO 16),
	CH505.M11 gp160 (aa SEQ ID NO 17; nt SEQ ID
	NO 18)
CH505w020.14	CH505w020.14D8gp120 (aa SEQ ID NO 19; nt SEQ
	ID NO: 20), CH505w020.14gp145 (nt SEQ ID NO
	21), CH505w020.14 gp160 (aa SEQ ID NO 22; nt
	SEQ ID NO 23)
CH505w030.28	CH505w030.28D8gp120 (aa SEQ ID NO 24; nt SEQ
	ID NO 25), CH505w030.28gp145(nt SEQ ID NO 26),,
	CH505w030.28 gp160 (aa SEQ ID NO 27; nt SEQ ID
	NO 28)
CH505w078.15	CH505w078.15D8gp120 (aa SEQ ID NO 29; nt SEQ
	ID NO 30), CH505w078.15gp145 (nt SEQ ID NO
	40), CH505w078.15 gp160 (aa SEQ ID NO 41; nt
	SEQ ID NO 42)
CH505w053.31	CH505w053.31D8gp120 (aa SEQ ID NO 43; nt SEQ
	ID NO 44), CH505w053.31gp145 (nt SEQ ID NO
	45), CH505w053.31 gp160 (aa SEQ ID NO 46; nt
	SEQ ID NO 47)
CH505w030.21	CH505w030.21D8gp120 (aa SEQ ID NO 48; nt SEQ
	ID NO 49), CH505w30.21gp145 (nt SEQ ID NO
	50), CH505w030.21 gp160 (aa SEQ ID NO 51 nt
	SEQ ID NO 52)
CH505w078.33	CH505w78.33gp120 (aa SEQ ID NO 54; nt SEQ ID
	NO 55), CH505w78.33gp145 (nt SEQ ID NO 56),
	CH505w078.33 gp160 (aa SEQ ID NO 57; nt SEQ
	ID NO 58)
CH505w053.16	CH505w053.16D8gp120 (aa SEQ ID NO 59; nt SEQ
	ID NO 60), CH505w53.16gp145 (nt SEQ ID NO
	61), CH505w053.16 gp160 (aa SEQ ID NO 62; nt
	SEQ ID NO 63)
CH505w100.B6	CH505w100.B6D8gp120 (aa SEQ ID NO 64; nt SEQ
	ID NO 65), CH505w100.B6gp145 (nt SEQ ID NO
	66), CH505w100.B6 gp160 (aa SEQ ID NO 67; nt
	SEQ ID NO 68)

HIV-1 Envelopes Selection E: Ten Early Envelopes (FIG. 24)


FIG. 24	HIV-1 Envelopes Selection E

CH505M11gp160 (aa SEQ ID NO 69)	CH505.M11
CH505w004.03gp160 (aa SEQ ID NO 70)	CH505.w004.03
CH505w020.14gp160 (aa SEQ ID NO 71)	CH505.w020.14
CH505w030.28gp160 (aa SEQ ID NO 72)	CH505.w030.28
CH505w30.12gp160 (aa SEQ ID NO 73)	CH505.w030.12
CH505w020.2gp160 (aa SEQ ID NO 74)	CH505.w020.2
CH505w030.10gp160 (aa SEQ ID NO 75)	CH505.w030.10
CH505w078.15gp160 (aa SEQ ID NO 76)	CH505.w078.15
CH505w030.19gp160 (aa SEQ ID NO 77)	CH505.w030.19
CH505w030.21gp160 (aa SEQ ID NO 78)	CH505.w030.21

HIV-1 Envelopes Selection C: Four Envelopes (FIG. 22)


FIG. 22	HIV-1 Envelopes Selection C

CH505w000.TFgp160 (aa SEQ ID NO 79)	703010505.TF,
CH505w053.16gp160 (aa SEQ ID NO 80)	703010505.W53.16
CH505w078.33gp160 (aa SEQ ID NO 81)	703010505.W78.33
CH505w100.B6gp160 (aa SEQ ID NO 82)	703010505.W100.B6

HIV-1 Envelopes Selection F: Ten Production Envelopes (10PR)

	TABLE 5

	FIG. 17

CH505.T/F	CH505w000.TFgp160 (aa SEQ ID NO 79) from
	FIG. 22
CH505.M11	CH505.M11D8gp120 (aa SEQ ID NO 14; nt SEQ
	ID NO 15), CH505.M11gp145 (nt SEQ ID NO 16),
	CH505.M11 gp160 (aa SEQ ID NO 17; nt SEQ
	ID NO 18)
CH505w020.14	CH505w020.14D8gp120 (aa SEQ ID NO 19; nt SEQ
	ID NO: 20), CH505w020.14gp145 (nt SEQ ID NO
	21), CH505w020.14 gp160 (aa SEQ ID NO 22; nt
	SEQ ID NO 23)
CH505w030.28	CH505w030.28D8gp120 (aa SEQ ID NO 24; nt SEQ
	ID NO 25), CH505w030.28gp145(nt SEQ ID NO 26),,
	CH505w030.28 gp160 (aa SEQ ID NO 27; nt SEQ
	ID NO 28)
CH505w078.15	CH505w078.15D8gp120 (aa SEQ ID NO 29; nt SEQ
	ID NO 30), CH505w078.15gp145 (nt SEQ ID NO 40),
	CH505w078.15 gp160 (aa SEQ ID NO 41; nt SEQ
	ID NO 42)
CH505w053.31	CH505w053.31D8gp120 (aa SEQ ID NO 43; nt SEQ
	ID NO 44), CH505w053.31gp145 (nt SEQ ID NO 45),
	CH505w053.31 gp160 (aa SEQ ID NO 46; nt SEQ
	ID NO 47)
CH505w030.21	CH505w030.21D8gp120 (aa SEQ ID NO 48; nt SEQ
	ID NO 49), CH505w30.21gp145 (nt SEQ ID NO 50),
	CH505w030.21 gp160 (aa SEQ ID NO 51; nt SEQ
	ID NO 52)
CH505w078.33	CH505w78.33gp120 (aa SEQ ID NO 54; nt SEQ ID
	NO 55), CH505w78.33gp145 (nt SEQ ID NO 56),
	CH505w078.33 gp160 (aa SEQ ID NO 57; nt SEQ
	ID NO 58)
CH505w053.16	CH505w053.16D8gp120 (aa SEQ ID NO 59; nt SEQ
	ID NO 60), CH505w53.16gp145 (nt SEQ ID NO 61),
	CH505w053.16 gp160 (aa SEQ ID NO 62; nt SEQ
	ID NO 63)
CH505w100.B6	CH505w100.B6D8gp120 (aa SEQ ID NO 64; nt SEQ
	ID NO 65), CH505w100.B6gp145 (nt SEQ ID NO
	66), CH505w100.B6 gp160 (aa SEQ ID NO 67; nt
	SEQ ID NO 68)

Claims

What is claimed is:

1. A composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.M6, CH505.M11, CH505w020.14, CH505w030.28, CH505w078.15, CH505w053.31, CH505w030.21, CH505w078.33, CH505w053.16, CH505w100.B6 or a combination thereof, or polynucleotide encoding the same.

2. A composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.T/F; CH505.M11; CH505w020.14; CH505w030.28; CH505w030.21; CH505w053.16; CH505w053.31; CH505w078.33; CH505w078.15; CH505w100.B6 or a combination thereof, or a polynucleotide encoding the same.

3. A composition comprising any one of the HIV-1 envelope polypeptides corresponding to the HIV-1 envelopes CH505.M11, CH505.w004.03, CH505.w020.14, CH505.w030.28, CH505.w030.12, CH505.w020.2, CH505.w030.10, CH505.w078.15, CH505.w030.19 CH505.w030.21 or a combination thereof, or polynucleotide encoding the same.

4. The composition of claims 1-3, wherein each HIV-1 envelope polypeptide comprises polypeptide which is a gp 41, gp 120, gp 145, gp 150 or gp 160 variant.

5. The composition of claims 1-4, wherein the HIV-1 envelopes further comprise a peptide or polynucleotides corresponding to a trimerization domain selected from a group consisting GCN4 and CD40L.

6. The composition of claim 5, wherein the trimerization domain is linked to the envelope sequence by an amino acid linker 3-20 amino acids long.

7. A composition comprising MPER-peptide-liposome-CD40L conjugate.

8. The composition of claim 7, further comprising an N-terminal histone tag.

9. The composition of any one of claims 1-8 further comprising an adjuvant.

10. A method of inducing an immune response in a subject comprising administering the composition of any one of claims 1-9 in an amount sufficient to induce an immune response.

11. The method of claim 10, further comprising administering chloloquine before each immunization.

12. The method of claim 10, further comprising administering anti-CD25 antibody before each immunization.

13. The method of claim 10, further comprising administering anti-CD25 antibody after each immunization.

14. The method of claim 10, wherein the composition comprises a nucleic acid, a protein or any combination thereof.

15. The method of claim 14, wherein the nucleic acid encoding the envelope is operably linked to a promoter inserted in an expression vector.

16. The method of claim 14, wherein the protein is recombinant.

17. The method of claim 14, wherein the composition is administered as a prime, a boost, or both.

18. The method of claim 14, wherein the composition is administered as a multiple boosts.