WO2017139392A1

WO2017139392A1 - Recombinant hiv-1 envelope proteins and their use

Info

Publication number: WO2017139392A1
Application number: PCT/US2017/017038
Authority: WO
Inventors: Paolo Lusso; Qingbo LIU
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2016-02-08
Filing date: 2017-02-08
Publication date: 2017-08-17

Abstract

Recombinant gp120 proteins, recombinant HIV-1 Env ectodomain trimers, and isolated peptides including gp120 sequences are disclosed, as well as nucleic acids encoding and methods of producing such proteins and peptides. Methods are also provided for the treatment or prevention of a human immunodeficiency type 1 (HIV-1) infection. The methods can include administering to a subject a recombinant gp120, recombinant HIV-1 Env ectodomain trimer, or isolated peptide as disclosed herein. In some embodiments, administering a recombinant gp120, recombinant HIV-1 Env ectodomain trimer, or isolated peptide as disclosed herein to a subject elicits an immune response to HIV-1 in the subject.

Description

RECOMBINANT HIV-1 ENVELOPE PROTEINS AND THEIR USE

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/292,750, filed February 8, 2016, which is incorporated by reference in its entirety.

FIELD

This disclosure relates to recombinant Human Immunodeficiency Virus type 1 (HIV-1) gpl20, HIV- 1 Envelope (Env) ectodomain trimers including the recombinant gpl20, and isolated peptides for treatment and prevention of HIV-1 infection and disease.

BACKGROUND

Millions of people are infected with HIV-1 worldwide, and 2.5 to 3 million new infections have been estimated to occur yearly. Although effective antiretroviral therapies are available, millions succumb to AIDS every year, especially in sub-Saharan Africa, underscoring the need to develop measures to prevent the spread of this disease.

An enveloped virus, HIV-1 hides from humoral recognition behind a wide array of protective mechanisms. The major envelope protein of HIV-1 is a glycoprotein of approximately 160 kD (gpl60). During infection, proteases of the host cell cleave gpl60 into gpl20 and gp41. Gp41 is an integral membrane protein, while gpl20 protrudes from the mature virus. Together gpl20 and gp41 make up the HIV-1 envelope spike, which is a target for neutralizing antibodies.

It is believed that immunization with an effective immunogen based on HIV-1 Env can elicit a neutralizing response, which may be protective against HIV-1 infection. Further, it is believed that peptide therapeutics that bind gpl20 can neutralize its function, and thus inhibit HIV- 1 infection. However, despite extensive effort, a need remains for agents capable of such action.

SUMMARY

Disclosed herein is the surprising finding that the CD4 binding site on the trimeric HIV-1 Env ectodomain (including gpl20 and the gp41 ectodomain) adopts a quarternary conformation including residues from multiple protomers of the ectodomain when the ectodomain is in the prefusion mature closed conformation. In addition to the "classic" CD4 binding site residues on the gpl20 outer domain ("CD4- BS 1"), CD4 binds to residues of the neighboring protomer including residues of the -1 helix (e.g., E62, T63, E64, H66) and β3-β4 loop (e.g., K207) of the gpl20 inner domain ("CD4-BS2"), as well as residues in the 550-570 segment of the neighboring gp41 protomer (e.g., E560, Q562, Q563, H564). CD4-interaction with the CD4-BS2 improves the stability of the CD4-gpl20 interaction, and triggers gpl20 conformational changes that enable coreceptor binding and progression of the fusogenic process. Elucidation of the complete CD4 binding site (including the CD4-BS 1 and CD4-BS2) on the HIV-1 Env trimer allowed for design of recombinant gpl20 proteins and HIV-1 Env ectodomain trimers that include stabilized forms of the complete CD4 binding site. Provided herein are recombinant gpl20 proteins including one or more amino acid substitutions that stabilize the CD4 binding site in a prefusion mature closed conformation, and/or that stabilize the CD4 binding site in a conformation comprising surface exposure of CD4-BS2 residues that contact CD4, such as gpl20 residues 62, 64, 66 and 207 (HXB2 numbering). The recombinant gpl20 or a HIV-1 Env ectodomain trimer including the recombinant gpl20 can be used to elicit an immune response to HIV-1 in a subject. In some embodiments, the elicited immune response can lead to increased production of broadly neutralizing CD4-binding site specific antibodies compared to HIV-1 immunogens that lack a stabilized CD4-BS2.

In some embodiments, an immunogen is provided that comprises a recombinant HIV-1 gpl20 comprising a non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109- 113 (HXB2 numbering). For example, the non-native disulfide bond can comprise a disulfide bond between cysteine residues introduced by A70C and LI 11C substitutions in the gpl20. In some embodiments, an immunogen is provided that comprises an amino acid substitution at gpl20 position 61 (HXB2 numbering), for example, a Y61A or a Y61F substitution. In some embodiments, an immunogen is provided that comprises mutation of a glycan sequon at position N262 and/or N301. For example, the glycan sequon at position N262 can be mutated by including N262Q and/or S264A substitutions in the recombinant HIV-1 gpl20, and the glycan sequon at position N301 can be mutated by including N301Q and/or T303A substitutions in the recombinant HIV-1 gpl20. The recombinant gpl20 comprises a CD4 binding site and specifically binds to CD4.

In some embodiments, the recombinant gpl20 can be a monomer.

In additional embodiments, the immunogen can comprise a recombinant HIV-1 Env ectodomain trimer comprising three gpl20-gp41 protomers comprising the recombinant gpl20 and a gp41 ectodomain.

In some such embodiments, the recombinant HIV-1 Env ectodomain trimer can be stabilized in a prefusion mature closed conformation by additional modifications, for example, by incorporation of one or more stabilizing mutations, such as a non-natural disulfide bond between cysteine substitutions at HIV-1 gp l20 positions 201 and 433 (e.g., I201C and A433C substitutions), a non-natural disulfide bond between cysteine substitutions at positions gp41 positions 501 and 605 (for example, A501C and T605C substitutions), and/or a proline substitution at gp41 position 559 (for example, an I559P substitution).

In additional embodiments, nucleic acid molecules encoding the recombinant gpl20 or HIV-1 Env ectodomain trimer, as well as vectors (such as an inactivated or attenuated viral vector) including the nucleic acid molecules are also provided.

Compositions including the disclosed immunogens are also provided. The composition may be a pharmaceutical composition suitable for administration to a subject, and may also be contained in a unit dosage form. The compositions can further include an adjuvant. The recombinant gpl20 or HIV-1 Env ectodomain trimer may also be conjugated to a carrier to facilitate presentation to the immune system. Methods of generating an immune response to HIV-1 Env in a subject are disclosed, as are methods of treating, inhibiting or preventing an HIV-1 infection in a subject. In such methods a subject, such as a human subject, is administered an effective amount of a disclosed immunogen to elicit the immune response. The subject can be, for example, a human subject at risk of or having an HIV-1 infection.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the

accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIGs. 1A-1C are a set of diagrams of the structure of the HIV-1 Env ectodomain trimer bound to

CD4 that illustrate the quaternary configuration of the CD4-binding site in the HIV-1 Env trimer (FIGs. 1A and IB), and a set of graphs illustrating gpl20 mutations that abrogate HIV-1 infectivity (FIG. 1C). FIG. 1A: Surface representation of 2-domain soluble CD4 (sCD4) docked onto the structure of a soluble HIV-1 Env trimer. Docking was performed by aligning the structure of a CD4-bound monomeric gpl20 inclusive of the N- terminal region (PDB ID: 3JWD) to the structure of one gpl20 protomer from the structure of a soluble cleaved BG505-SOSIP.664 trimer (PDB ID: 4TVP); the monomeric gpl20 was then hidden. The model shows that CD4 reaches deep into the inter-protomer groove and makes contact with two neighboring gpl20 protomers. FIG. IB: Close-up representation of the portion of the CD4-binding site found on the protomer adjacent to the "classic" CD4 binding site, refered to herein as the "CD4-BS2" domain of gpl20. Four residues in the CI ( -1 helix) and C2 (β3-β4 loop) regions of a neighboring gpl20 protomer positioned at less than 5 A from the docked sCD4 molecule are shown by stick representation. FIG. 1C: Disruption of CD4-BS2 abrogates HIV-1 infectivity. Infectious pseudoparticles were produced in HEK 293T cells by co-transfecting plasmids expressing wild-type (WT) and mutated BG505-T332N (clade A) or BaL (clade B) gpl60 together with a backbone plasmid (pSG3^Aenv) expressing a full-length HIV-1 clone with a defective Env gene. The infectivity was determined in TZM-bl cells by a luciferase assay. The pseudovirus input used for each mutant was normalized based on the concentration of p24cAC antigen in the pseudovirus stocks. Relative infectivity values for the mutants were calculated as percent of the value obtained with the WT pseudotypes, which was set at 100. Both individual and combined charge inversions of key residues in CD4-BS2 (H66, K207 and E62/E64) abrogated infectivity mediated by both envelopes.

FIGs. 2A-2E are a set of graphs showing that disruption of the CD4-BS2 reduces the stability of

CD4 binding and prevents the adoption of coreceptor-binding competence. FIG. 2A, Charge inversions in CD4-BS2 reduce the CD4-binding capacity of a soluble cleaved HIV-1 Env trimer. Purified WT and mutated BG505-SOSIP.664 trimers were tested for binding to 4-domain sCD4 in ELISA. The trimers were captured using mAb 2G12 and all values were normalized relative to binding of a rabbit anti-His tag antiserum; binding of sCD4 was revealed using mAb OKT4. The correct folding of the trimers was evaluated by reactivity with a large panel of anti-gpl20 mAbs (see FIGs. 9A and 9B). FIG. 2B, Charge inversions in CD4-BS2 reduce binding of soluble cleaved trimers to native CD4 expressed on the surface of PM1 cells. The specificity of CD4 binding was demonstrated by abrogation of the signal upon preincubation of the trimers with sCD4. Binding of the trimer was evaluated by flow cytometry using mAb 2G12. FIG. 2C, Kinetics of sCD4 binding to the WT and mutated trimers measured by single-cycle surface plasmon resonance (SPR). MAb 2G12 was immobilized on the sensor chip to capture the trimers; sCD4 was injected in the flow phase. Binding curves were fitted globally to a 1 : 1 Langmuir model. FIG. 2D, Charge inversions in CD4-BS2 abrogate the CCR5-binding capacity of BG505-SOSIP.664 trimers after sCD4 treatment. Binding of WT and mutated soluble BG505-SOSIP.664 trimers to CCR5 expressed on the surface of Cf2Th/syn-CCR5 cells was assessed by flow cytometry. The trimers were treated with or without 2-domain sCD4, and then incubated with Cf2Th/syn-CCR5 cells. An anti-His tag mAb was used to detect the cell surface-bound trimers. The mean fluorescence intensity (MFI) values are presented. Only the sCD4- treated WT trimer effectively bound to CCR5, whereas all the CD4-BS2 mutants, as well as the control mutant D368R, did not exhibit significant binding regardless of sCD4 treatment. The specificity of binding to CCR5 was assessed by complete abrogation of the signal upon treatment of the cells with an anti-CCR5 mAb. FIG. 2E, Binding of mAb 48d to WT and mutated soluble BG505-SOSIP.664 trimers, as determined by ELISA. WT or mutated trimers were captured on the plate by rabbit anti-His tag antiserum and treated with or without sCD4 before the addition of 48d, an anti-gpl20 mAb recognizing an epitope that widely overlaps the CCR5-binding site (Xiang et al., AIDS Res. Hum. Retroviruses, 18, 1207-1217, 2002).

Normalized values were calculated as percent of those obtained with the reference mAb 2G12.

FIGs. 3A and 3B are a ribbon diagram and a set of graphs illustrating that disruption of the CD4- BS2-interactive site reduces the HIV-1 receptor function of CD4. FIG. 3A, Surface representation of 2- domain sCD4 docked onto the structure of the BG505-SOSIP.664 HIV-1 Env trimer. Docking was performed as described in FIGs. 1A- 1C. In the magnified inset, the CD4 contact sites with CD4-BS2 predicted by docking and MD simulation (see Table 2 in Example 1) are highlighted by stick representation. FIG. 3B, Infectivity of pseudoviruses carrying the WT BG505-T332N or BaL envelopes in cells expressing different CD4 mutants. Luciferase-expressing pseudoviruses were produced in 293T cells by co-transfecting plasmids expressing BG505-T332N or BaL gpl60 together with a backbone plasmid (pNL4-3.Luc.R^~E^~) expressing full-length HIV- 1 clone carrying the firefly luciferase gene and two defective genes (Env and Vpr). Cf2Th/syn-CCR5 cells transfected with plasmids encoding WT or mutated full-length CD4 were used as targets in single-round infection assays. Charge inversions of E13, K22, or D63 in CD4 caused dramatic reductions of HIV-1 infectivity while K21 had limited effects. Alanine substitution of F43 was tested as a control. Relative infectivity values for the mutants were calculated as percent of the value obtained with WT CD4.

FIGs. 4A and 4B are a set of ribbon diagrams and graphs illustrating that disruption of CD4-BS2 affects binding of neutralizing monoclonal antibodies specific for the CD4-binding site. FIG. 4A, Surface representation of selected neutralizing mAbs specific for the CD4-binding site docked onto the HIV-1 Env trimer. The structure of each antibody-bound monomeric gpl20 was aligned to one protomer of the BG505- SOSIP.664 trimer structure (PDB ID: 4TVP), followed by hiding of the monomeric gpl20. All the mAbs were found to reach deep into the inter-protomer groove and in some cases (i.e., VRC03, VRC06, VRC- CH31) to make direct contact with CD4-BS2. A close-up view of the putative contact area with CD4-BS2 is shown below each docked trimer, with key residues (E62, E64, H66, K207) highlighted by stick representation. FIG. 4B, Binding of selected anti-CD4-BS mAbs to WT and mutated soluble cleaved HIV-1 Env trimers (BG505-SOSIP.664), as assessed by ELISA. A rabbit anti-His tag antibody was used to capture the trimers. Normalized values were calculated as percent of those obtained with the reference mAb 2G12.

FIGs. 5A and 5B are a set of schematic diagrams illustrating the quaternary configuration of the CD4-binding site and amino acid conservation within the CD4-BS2. FIG. 5A, Surface representation of the complete CD4-binding site modeled in the crystal structure of a soluble cleaved HIV-1 Env trimer (BG505- SOSIP.664). In the quaternary structure, CD4 makes contact with two neighboring protomers (colored in dark and light gray, respectively). FIG. 5B, Amino acid conservation within the CD4-BS2 region.

Sequence alignment of a segment of the CI region (centered around the -1 helix) and of the C2 region (a segment of the β3-β4 loop) of the gpl20 inner domain from the all the available group-M HIV-l/SIVcpz isolates (including both subtypes A-K and recombinant forms) was obtained from the Los Alamos HIV database. E64, H66 and K207 are nearly universally conserved (>99.7%) across all group-M HIV-1 isolates; position 62 is occupied by an acidic residue (either E or D) in more than 90% of group-M isolates, with non-conservative mutations essentially limited to genetic subtype D (58% E^K) and subtypes G and J (100% E^S). The height of each stack indicates the degree of conservation for each residue; the relative height of each letter within individual stacks represents the frequency of the indicated amino acid at that site.

FIGs. 6A and 6B are a set of graphs illustrating the reactivity of wild- type and mutated gpl60 expressed on the surface of transfected cells with anti-envelope monoclonal antibodies. WT and mutated BG505-T332N (clade A) or BaL (clade B) gpl60 were expressed on the surface membrane of HEK 293T cells and tested for their reactivity with various anti-envelope mAbs by flow cytometry. Of note, the trimer - preferring mAbs PG9, 35022 and PGT151 showed similar binding levels to the WT and the mutants, indicating a correct folding and trimeric configuration for both envelopes. As expected, the anti-CD4-BS mAb VRCOl did not react with the D368R mutant. MFI values were normalized relative to the MFI obtained with the reference mAb 2G12.

FIGs. 7A and 7B are a set of graphs illustrating the effect of CD4-BS2 mutations on gpl20-gp41 association. FIG. 7A, WT and mutated BG505-T332N or BaL gpl60 were expressed on the surface membrane of HEK 293T cells and supernatants were collected on day 2 and day 4 for testing spontaneous gpl20 shedding by ELISA. MAb 2G12 was used to capture shed gpl20s from the supernatants, and the signal was revealed by anti-SF2. FIG. 7B, The level of expression of gpl60 on the cell membrane was evaluated by flow cytometry using mAbs 2G12, PG9 and 2F5.

FIG. 8 is a graph illustrating the effect of individual and combined mutations in the inner domain of gpl20 on infectivity of viral pseudotypes carrying the HIV-1 BaL envelope. Individual and combined mutations both within and in the proximity of CD4-BS2 were introduced into the BaL gpl60 glycoprotein. Viral pseudotypes carrying the WT or mutated BaL gpl60 were generated and tested for infectivity in TZM- bl cells by luciferase assay. Relative infectivity values for the mutants were calculated as percent of the value obtained with the WT pseudotypes, which was set at 100.

FIGs. 9A and 9B are a set of graphs illustrating the reactivity of wild-type and mutated soluble cleaved HIV-1 Env ectodomain trimers (SOSIP BG505-SOSIP.664) with anti-gpl20 monoclonal antibodies. A panel of anti-gpl20 antibodies was used to verify the correct folding of purified WT and mutated SOSIP BG505-SOSIP.664 trimers expressed in HEK 293FS cells by ELISA. His-tagged trimers were captured using a rabbit anti-His tag antibody directly coated on the plate. Of note, mAb F105, which reacts well with monomeric gpl20 but not with the functional trimer, was virtually negative on all trimers, whereas trimer- preferring mAbs (eg, PG9, PGT145, PGT151) showed similar binding levels to the wild-type and the mutants, indicating the correct folding of the trimers. As expected, anti-CD4-BS mAbs (eg, VRCOl,

VRC07, 3BNC117) did not react significantly with the D368R mutant. Optical density (OD) values were normalized relative to the level of binding obtained with the reference mAb 2G12.

FIGs. lOA-lOC are a set of graphs illustrating the effect of mutations in the CD4-BS2 on monomeric gpl20 binding to CD4. FIG. 10A, Binding of sCD4 to WT and mutated monomeric gpl20 from BG505-T332N and BaL, as determined by ELISA. The values were normalized relative to binding of a polyclonal anti-gpl20 antiserum (anti-SF2). FIGs. 10B and IOC, Reactivity of wild-type and mutated monomeric gpl20 with anti-gpl20 monoclonal antibodies. Monomeric gpl20 from two different strains (BG505-T332N, clade A, and BaL, clade B) and respective mutants were expressed in HEK 293FS cells and tested with various anti-gpl20 mAbs in ELISA. Each gpl20 was captured by a plastic-coated hyperimmune anti-gpl20 C-terminus antibody (D7324). These data confirmed the correct folding of the mutants showing reactivity with all the reference anti-gpl20 mAbs tested, with the exception of VRCOl and bl2 with mutant D368R, as expected. Optical density (OD) values were normalized relative to the level of binding obtained with the reference mAb 2G12.

FIG. 11 is a set of diagrams showing the spatial orientation of CD4-BS2 residues in the crystal structures of trimeric and monomeric gpl20. Stick representation of the key residues in CD4-BS2 in the structure of a BG505-SOSIP.664 trimer (PDB ID: 4VTP) and a gpl20 monomer (PDB ID: 3JWD). A side view is shown in the upper panels, with the side chains of key residues highlighted by stick representation; a front view surface representation is shown in the lower panels. In trimeric structure, CD4-BS2 forms a continuous ridge of electrostatically-charged surface, with the side chains of K207, E62, E64 and H66 well exposed to the solvent; in contrast, in monomeric gpl20, they are partially or completely buried. Of note, the spatial orientation of these residues is nearly identical in all the available monomeric gpl20 structures, regardless of the presence and nature of co-crystallized ligands (PDB ID: 3TGQ, 3TGR, 3TGS, 3TGT, 3TIH, 3JWD, 3JWO, 4LSS, 4LSU, 4J6R, 4LSP, 3SE8, 4JB9, 3SE9, 4JDT, 4JPV, 4JPW, 4JZZ).

FIGs. 12A and 12B are a set of graphs showing that mutations in the CD4-BS2-contact site of CD4 affect HIV-1 entry. FIG. 12A, Mutations were introduced in full-length human CD4 and the mutants were expressed in Cf2Th/syn CCR5 target cells. Luciferase-expressing pseudoviruses carrying WT BaL or BG505-T332N envelopes were used for infectivity assay. Mutations of residues F43 and R59, which contact the classic CD4-BS in gpl20, were used as controls. Relative infectivity values for the mutants were calculated as percent of the value obtained with WT CD4. FIG. 12B, Reactivity of WT and mutated CD4 with anti-CD4 monoclonal antibodies. Full-length WT or mutated human CD4 was expressed on the surface membrane of HEK 293T cells and tested using anti-CD4 mAbs by flow cytometry. MFI values were normalized relative to the MFI obtained with the reference mAb OKT4, which targets domain 3 of CD4, whose reactivity is not affected by mutations within domain 1.

FIGs. 13A and 13B are a set of graphs showing binding data of VRC03 and VRC06 to HIV-1 trimers and monomers. FIG. 13A, SPR analysis of VRC03 and VRC06 binding to WT and mutated soluble cleaved HIV-1 Env trimers (BG505-SOSIP.664). Immobilized anti-His tag antibody was used to capture purified WT and mutated BG505-SOSIP.664 trimers to 400-500 RU. To assess binding, anti-gpl20 mAbs were injected at 25 μg/ml (VRC03) or 50 μg/ml (VRC06) followed by a dissociation phase of 2 min.

Surface regeneration was performed after each round of injection. Binding curves were recorded over time and corrected by extracting both control cell and buffer-injection responses. FIG. 13B, Lack of reactivity of VRC03 and VRC06 with monomeric gpl20. Binding of selected anti-CD4-BS mAbs to WT monomeric gpl20 was assessed by ELISA. gpl20 from BG505-T332N or BaL were captured using an anti-gpl20 C- terminus hyperimmune serum, D7324, directly coated on the plate. All of the values were normalized against binding of the reference antibody 2G12. As expected, VRCOl reactivity was abrogated by the D368R mutation.

FIGs. 14A-14D are a set of graphs illustrating the reactivity of wild-type and mutated gpl60 expressed on the surface of transfected cells with monoclonal antibodies against the CD4-binding site. A wide panel of human mAbs directed to the CD4-BS was tested for binding to the WT and mutated HIV-1 BG505-T332N (FIGs. 14A and 14B) or BaL (FIGs. 14C and 14D) gpl60 expressed on the surface of HEK 293T cells. Binding levels were determined by flow cytometry. MFI values were normalized relative to the MFI obtained with the reference mAb 2G12.

FIG. 15 is a set of graphs showing results from rabbit immunization assays using a peptide including SEQ ID NO: 20. The sera obtained from immunized rabbits were tested against the immunizing homologous peptide as well as against recombinant homologous gpl20-BaL or heterologous BG505-SOSIP trimer by ELISA. SEQUENCES

The nucleic and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file named "Sequence.txt" (-132 kb), which was created on February 5, 2017, and which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NOs: 1-9 are the amino acid sequences of exemplary HIV-1 Env proteins. SEQ ID NOs: 10-11 are the amino acid sequences of furin cleavage sites.

SEQ ID NO: 12 is the amino acid sequence of a peptide linker.

SEQ ID NOs: 13-15 are the amino acid sequences of transmembrane domains.

SEQ ID NO: 16 is the amino acid sequence of a T4 fibritin trimerization domain.

SEQ ID NOs: 17-20 are the amino acid sequences of immunogenic HIV-l Env peptides.

SEQ ID NOs: 21-29 are the amino acid sequences of exemplary HIV-l Env proteins containing or more modifications for stabilization in a prefusion mature closed conformation.

DETAILED DESCRIPTION

I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley- VCH in 16 volumes, 2008; and other similar references.

As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes single or plural antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. In some embodiments, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water - in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages).

Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a "biological adjuvant"), such as costimulatory molecules.

Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-lBBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. In some embodiments, the Adjuplex™ (Advanced BioAdjuvants) can be used with a disclosed recombinant gpl20, or an HIV- l Env ectodomain trimer including the recombinant gpl20 to elicit an immune response to HIV-l Env. Additional description of Adjuvants can be found, for example, in Singh ((ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed

immunogens.

Administration: The introduction of a composition into a subject by a chosen route.

Administration can be local or systemic. For example, if the chosen route is intravenous, the composition (such as a composition including a disclosed immunogen) is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitutions: The replacement of one amino acid in a polypeptide with a different amino acid or with no amino acid (i.e., a deletion). In some examples, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide, for example, an amino acid in a recombinant Clade A HIV-1 Env polypeptide can be substituted with the corresponding amino acid from a Clade B HIV-1 Env polypeptide.

Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), such as HIV-1 gpl20, an antigenic fragment thereof, or a dimer or multimer of the antigen. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen- binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g.,

Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010). Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al. , Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen.

CD4: Cluster of differentiation factor 4 polypeptide; a T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV-1 on T- cells during HIV-1 infection. CD4 is known to bind to gpl20 from HIV-1 Env. The sequence of the CD4 precursor has a hydrophobic signal peptide, an extracellular region of approximately 370 amino acids, a highly hydrophobic stretch with significant identity to the membrane-spanning domain of the class II MHC beta chain, and a highly charged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985). The amino acid sequence of human CD4 is deposited in GenBank as No. P01730.1. Several embodiments utilize sCD4, which includes the extracellular domain of CD4 (without the signal peptide), approximately CD4 amino acids 26- 390. sCD4 can be obtained commercially (e.g., from Mybiosource); methods of its production are well known in the art.

Control: A reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In other embodiments, the control is a positive control sample obtained from a patient diagnosed with HIV-1 infection. In still other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of HIV-1 patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example, a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

Degenerate variant: In the context of the present disclosure, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as a disclosed recombinant gpl20) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

Expression: Transcription or translation of a nucleic acid sequence. For example, a gene is expressed when its DNA is transcribed into an RNA or RNA fragment, which in some examples is processed to become mRNA. A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. The term "expression" is used herein to denote either transcription or translation. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

Expression control sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell- type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originated from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a disclosed recombinant gpl20 is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example, transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

Glycosylation site: An amino acid sequence on the surface of a polypeptide, such as a protein, which accommodates the attachment of a glycan. An N-linked glycosylation site is triplet sequence of NX(S/T) in which N is asparagine, X is any residues except proline, and (SIT) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

Human Immunodeficiency Virus Type 1 (HIV-1): A retrovirus that causes immunosuppression in humans (HIV-1 disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). "HIV-1 disease" refers to a well -recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV-1 virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease include a progressive decline in T cells. Related viruses that are used as animal models include simian

immunodeficiency virus (SIV), and feline immunodeficiency virus (FTV). Treatment of HIV-1 with HAART has been effective in reducing the viral burden and ameliorating the effects of HIV-1 infection in infected individuals.

HIV-1 envelope protein (Env): The HIV-1 Env protein is initially synthesized as a precursor protein of 845-870 amino acids in size, designated gpl60. Individual gpl60 polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately positions 511/512 to generate separate gpl20 and gp41 polypeptide chains, which remain associated as gpl20-gp41 protomers within the homotrimer. The ectodomain (that is, the extracellular portion) of the HIV-1 Env trimer undergoes several structural rearrangements from a prefusion mature (cleaved) closed conformation that evades antibody recognition, through intermediate conformations that bind to receptors CD4 and co-receptor (either CCR5 or CXCR4), to a postfusion conformation. The HIV-1 Env ectodomain includes the gpl20 (approximately HIV-1 Env positions 31-511) and the gp41 ectodomain (approximately HIV-1 Env positions 512-644). An HIV-1 Env ectodomain trimer includes a protein complex of three HIV-1 Env ectodomains.

Mature gpl20 includes approximated HIV-1 Env residues 31-511, contains most of the external, surface-exposed, domains of the HIV-1 Env trimer, and it is gpl20 which binds both to cellular CD4 receptors and to cellular chemokine receptors (such as CCR5). A mature gpl20 polypeptide is an extracellular polypeptide that interacts with the gp41 ectodomain to form an HIV-1 Env protomer that trimerizes to form the HIV-1 Env ectodomain trimer. The mature gpl20 wild-type polypeptide is heavily N- glycosylated, giving rise to an apparent molecular weight of 120 kD. Native gpl20 includes five conserved regions (C1-C5) and five regions of high variability (V1-V5). See FIG. 11 for an illustration of gpl20 primary and secondary structures.

Mature gp41 includes approximately HIV-1 Env residues 512-860, and includes cytosolic-, transmembrane-, and ecto-domains. The gp41 ectodomain (including approximately HIV-1 Env residues 512-644) can interact with gpl20 to form an HIV-1 Env protomer that trimerizes to form the HIV-1 Env trimer. The prefusion mature closed conformation of the HIV-1 Env ectodomain trimer is a structural conformation adopted by HIV-1 Env ectodomain trimer after cellular processing to a mature prefusion state with distinct gpl20 and gp41 polypeptide chains, and before specific binding to the CD4 receptor. The three-dimensional structure of an exemplary HIV-1 Env ectodomain trimer in the prefusion mature closed conformation is known (see, e.g., Pancera et al, Nature, 514:455-461, 2014). In the prefusion mature closed conformation, the HIV-1 Env ectodomain trimer includes a V1V2 domain "cap" at its membrane distal apex, with the V1V2 domain of each Env protomer in the trimer coming together at the membrane distal apex. At the membrane proximal aspect, the prefusion mature closed conformation of the HIV-1 Env ectodomain trimer includes distinct cc6 and cc7 helices. CD4 binding causes changes in the conformation of the HIV-1 Env ectodomain trimer, including disruption of the VlVl domain cap, which "opens" as each V1V2 domain moves outward from the longitudinal axis of the Env trimer, and formation of the HR1 helix, which includes both the cc6 and cc7 helices (which are no longer distinct). These conformational changes bring the N- terminus of the fusion peptide within close proximity of the target cell membrane, and expose "CD4- induced" epitopes (such as the 17b epitope) that are present in the CD4-bound open conformation, but not the mature closed conformation, of the HIV-1 Env ectodomain trimer.

The numbering used in the disclosed HIV-1 Env proteins and fragments thereof (such as a gpl20 and gp41) is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al, Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber et al, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety. In one example, an HIV-1 Env protein is from the BG505 strain of HIV, which is a Clade A HIV-1 virus isolated from a six- week old HIV-1 infected infant. The amino acid sequence of BG505 Env protein is known (see, e.g., GenBank accession no. ABA61516, incorporated by reference herein as present in the database on June 20, 2014), and set forth as SEQ ID NO: 2.

HIV-1 Env ectodomain trimer stabilized in a prefusion mature closed conformation: A HIV-1

Env ectodomain trimer having one or more amino acid substitutions, deletions, or insertions compared to a native HIV-1 Env sequence that provide for increased retention of the prefusion mature closed conformation upon CD4 binding compared to a corresponding native HIV-1 Env sequence. In some embodiments, the HIV-1 Env ectodomain trimer can include one or more cysteine substitutions that allow formation of a non- natural disulfide bond that stabilizes the HIV-1 Env ectodomain trimer in its prefusion mature closed conformation.

A HIV-1 Env ectodomain trimer stabilized in the prefusion mature closed conformation has at least 90% (such as at least 95% or at least 99%) reduced transition to the CD4-bound open conformation upon CD4 binding compared to a corresponding native HIV- 1 Env sequence. The "stabilization" of the prefusion mature closed conformation by the one or more amino acid substitutions, deletions, or insertions can be, for example, energetic stabilization (for example, reducing the energy of the prefusion mature closed conformation relative to the CD4-bound open conformation) and/or kinetic stabilization (for example, reducing the rate of transition from the prefusion mature closed conformation to the prefusion mature closed conformation). Additionally, stabilization of the HIV- 1 Env ectodomain trimer in the prefusion mature closed conformation can include an increase in resistance to denaturation compared to a corresponding native HIV-1 Env sequence.

Methods of determining if a HIV-1 Env ectodomain trimer is in the prefusion mature closed conformation are provided herein, and include (but are not limited to) negative stain electron microscopy and antibody binding assays using a prefusion mature closed conformation specific antibody, such as VRC26 or PGT145. Methods of determining if a HIV- 1 Env ectodomain trimer is in the CD4-bound open conformation are also provided herein, and include (but are not limited to) negative stain electron microscopy and antibody binding assays using a CD4-bound open conformation specific antibody, such as 17b, which binds to a CD4-induced epitope. Transition from the prefusion mature closed conformation upon CD4 binding can be assayed, for example, by incubating a HIV-1 Env ectodomain trimer of interest that is in the prefusion mature closed conformation with a molar excess of CD4, and determining if the HIV- 1 Env ectodomain trimer retains the prefusion mature closed conformation (or transitions to the CD4-bound open conformation) by negative stain electron microscopy analysis, or antigenic analysis.

HXB2 numbering system: A reference numbering system for HIV protein and nucleic acid sequences, using the HIV-1 HXB2 strain sequences as a reference for all other HIV-1 strain sequences. The person of ordinary skill in the art is familiar with the HXB2 numbering system, and this system is set forth in "Numbering Positions in HIV Relative to HXB2CG," Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety. Unless context indicates otherwise, the numbering used in HIV-1 polypeptides disclosed herein is relative to the HXB2 numbering scheme. For reference, the amino acid sequence of HIV-1 Env of HXB2 is set forth as SEQ ID NO: 1 (GENBANK® Accession No. K03455, incorporated by reference herein as present in the database on June 20, 2014).

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen- specific response"). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. "Priming an immune response" refers to pre-treatment of a subject with an adjuvant to increase the desired immune response to a later administered immunogenic agent. "Enhancing an immune response" refers to co-administration of an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant.

Immunogen: A protein or a portion thereof that is capable of eliciting an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen can lead to protective immunity and/or proactive immunity against a pathogen of interest. In some examples, an immunogen comprises a recombinant HIV-1 Env ectodomain trimer as disclosed herein.

Immunogenic composition: A composition comprising a disclosed immunogen, or a nucleic acid molecule or vector encoding a disclosed immunogen, that induces a measurable CTL response against the immunogen, or induces a measurable B cell response (such as production of antibodies) against the immunogen, when administered to a subject. In one example, an immunogenic composition is a pharmaceutical composition that includes a disclosed recombinant HIV-1 Env ectodomain trimer or immunogenic fragment thereof, that induces a measurable CTL response against HIV-1, or induces a measurable B cell response (such as production of antibodies) against a HIV-1. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this immunogen). For in vivo use, the immunogenic composition will typically include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Isolated: An "isolated" biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been "isolated" include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule. Non-limiting examples of peptide linkers include glycine-serine peptide linkers. Unless context indicates otherwise, reference to "linking" a first polypeptide and a second polypeptide (or to two polypeptides "linked" together) refers to covalent linkage by peptide bond, or (if a peptide linker is involved) covalent linkage of the first and second polypeptides to the N and C termini of a peptide linker. Thus, reference to a gpl20 polypeptide "linked" to a gp41 ectodomain by a peptide linker indicates that the gpl20 polypeptide and the gp41 ectodomain are linked to opposite ends of the peptide linker by peptide bonds. Typically, such linkage is accomplished using molecular biology techniques to genetically manipulate DNA encoding the first polypeptide linked to the second polypeptide by the peptide linker.

Native protein, sequence, or di-sulfide bond: A polypeptide, sequence or di-sulfide bond that has not been modified, for example, by selective mutation. For example, selective mutation to focus the antigenicity of the antigen to a target epitope, or to introduce a di-sulfide bond into a protein that does not occur in the native protein. Native protein or native sequence are also referred to as wild-type protein or wild-type sequence. A non-native di-sulfide bond is a disulfide bond that is not present in a native protein, for example, a di-sulfide bond that forms in a protein due to introduction of one or more cysteine residues into the protein by genetic engineering.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti- sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. "cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include

pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired anti-HIV-1 immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A "residue" refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. "Polypeptide" is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

In many instances, one or more polypeptides can fold into a specific three-dimensional structure including surface-exposed amino acid residues and non-surface-exposed amino acid residues. In some instances a protein can include multiple polypeptides that fold together into a functional unit. For example, the HIV-1 Env protein is composed of three gpl20-gp41 protomers that trimerize in to a multimeric protein. "Surface-exposed amino acid residues" are those amino acids that have some degree of exposure on the surface of the protein, for example such that they can contact the solvent when the protein is in solution. In contrast, non-surface-exposed amino acids are those amino acid residues that are not exposed on the surface of the protein, such that they do not contact solution when the protein is in solution. In some examples, the non-surface-exposed amino acid residues are part of the protein core.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In some embodiments, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.

Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Homologs and variants of protein of interest are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the protein.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, /. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al, Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al, Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

As used herein, reference to "at least 80% identity" (or similar language) refers to "at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence. As used herein, reference to "at least 90% identity" (or similar language)refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence.

Signal Peptide: A short amino acid sequence (e.g., approximately 18-25 amino acids in length) that directs newly synthesized secretory or membrane proteins to and through membranes (for example, the endoplasmic reticulum membrane). Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases after the polypeptide has crossed the membrane. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). Exemplary signal peptide sequences are set forth as residues 1-30 of SEQ ID NO: 1 (HXB2 Env signal peptide) and SEQ ID NO: 2 (BG505 Env signal peptide).

Specifically bind: When referring to the formation of an antibody: antigen protein complex, or a protein:protein complex, refers to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide (for example, a glycoprotein), in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an particular antibody or protein binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example, gpl20) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art. A first protein or antibody specifically binds to a target protein when the interaction has a KD of less than 10^"6 Molar, such as less than 10^"7 Molar, less than 10^"8 Molar, less than 10^"9, or even less than 10^"10 Molar.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non- human mammals. In an example, a subject is a human. In a particular example, the subject is a newborn infant. In an additional example, a subject is selected that is in need of inhibiting of an HIV-1 infection. For example, the subject is either uninfected and at risk of HIV-1 infection or is infected in need of treatment.

Therapeutically effective amount: A quantity of a specified agent, such as a disclosed immunogen or immunogenic composition, that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of a disorder or disease, for example, to prevent, inhibit, and/or treat HIV-1 infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease, such as HIV-1 infection. For instance, this can be the amount necessary to inhibit or prevent viral replication or to measurably alter outward symptoms of the viral infection. In general, this amount will be sufficient to measurably inhibit virus replication or infectivity.

In one example, a desired response is to inhibit or reduce or prevent HIV-1 infection. The HIV-1 infected cells do not need to be completely eliminated or reduced or prevented for the composition to be effective. For example, administration of a therapeutically effective amount of the agent can decrease the number of HIV-1 infected cells (or prevent the infection of cells) by a desired amount, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HIV- 1 infected cells), as compared to the number of HIV- 1 infected cells in the absence of the composition.

It is understood that to obtain a protective immune response against a pathogen can require multiple administrations of the immunogenic composition. Thus, a therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a protective immune response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses, for example, daily, during a course of treatment (such as a prime-boost vaccination treatment). However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

Transmembrane domain: An amino acid sequence that inserts into a lipid bilayer, such as the lipid bilayer of a cell or virus or virus-like particle. A transmembrane domain can be used to anchor an antigen to a membrane. In some examples a transmembrane domain is a HIV-1 Env transmembrane domain. Exemplary HIV-1 Env transmembrane domains are provided herein, for example, as SEQ ID NO: 13.

Treating or inhibiting a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as HIV-1 infection or acquired immunodeficiency syndrome (AIDS). "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

The term "reduces" is a relative term, such that an agent reduces a response or condition if the response or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Similarly, the term "prevents" does not necessarily mean that an agent completely eliminates the response or condition, so long as at least one characteristic of the response or condition is eliminated. Thus, an immunogenic composition that reduces or prevents an infection or a response, can, but does not necessarily completely, eliminate such an infection or response, so long as the infection or response is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% of (that is to 10% or less than) the infection or response in the absence of the agent, or in comparison to a reference agent.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of an immunogenic protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.

II. Immunogens

A. Recombinant gpl20 proteins and HIV-1 Env ectodomain trimers

As disclosed herein, in the prefusion mature closed conformation of the HIV-1 Env ectodomain trimer, the CD4 binding site adopts a quarternary conformation that includes gpl20 residues from multiple protomers within the trimer. In addition to the "classic" CD4 binding site on the gpl20 outer domain ("CD4-BS1 domain"), CD4 binds to residues of the neighboring protomer including residues of the -1 helix (e.g., E62, T63, E64, H66) and β3-β4 loop (e.g., K207) from the gpl20 inner domain "CD4-BS2." Moreover, CD4 makes contact with residues in the aa. 550-570 segment of the neighboring gp41 protomer (e.g., E560, Q562, Q563, H564). CD4 interaction with the CD4-BS2 facilitates stability of the CD4-gpl20 interaction, triggering of gpl20 conformational changes that enable coreceptor binding, and progression of the fusogenic process. In contrast, in the gpl20 monomer, the CD4-BS2 residues are occluded by neighboring amino acids, reducing their surface exposure and therefore their ability to bind CD4.

Elucidation of the complete CD4 binding site (including the CD4-BS 1 and CD4-BS2) on the HIV-1 Env trimer allowed for design of recombinant gpl20 proteins and HIV-1 Env ectodomain trimers including the recombinant gpl20 proteins that include stabilized forms of the complete CD4 binding site. For example, the recombinant gpl20 and/or recombinant HIV-1 Env ectodomain trimer including the recombinant gpl20 can include one or more amino acid substitutions that stabilize the CD4 binding site in a prefusion mature closed conformation, and/or that stabilize the CD4 binding site in conformation comprising surface exposure of gpl20 residues 62, 64, 66 and 207 (HXB2 numbering). The recombinant gpl20 and HIV-1 Env ectodomain trimers can be used to elicit an immune response to HIV-1 in a subject. In some examples, the elicited immune response can lead to increased production of broadly neutralizing CD4-binding site specific antibodies compared to HIV-1 immunogens that lack a stabilized CD4-BS2.

Thus, the recombinant gpl20 proteins and HIV-1 Env ectodomain trimers including such recombinant gpl20 proteins disclosed herein are useful to inhibit or treat HIV-1 infection, and/or elicit an immune response in vertebrate animals (such as mammals, for example, primates, such as humans) to HIV- 1. Methods of making and using such molecules are also disclosed.

In some embodiments, the recombinant gpl20 or HIV-1 Env ectodomain trimer includes one or more non-natural disulfide bonds between a pair of cysteines that stabilizes the CD4-BS2 of the recombinant gpl20 or HIV-1 Env ectodomain trimer in the prefusion mature closed conformation. The cysteine residues that form the non-native disulfide bond can be introduced into a native gpl20 sequence by one or more amino acid substitutions, or insertions.

In some embodiments, the recombinant gpl20 or HIV-1 Env ectodomain trimer can include a non- native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113 (HXB2 numbering). For example, the recombinant gpl20 can include a non-native disulfide bond between cysteine residues introduced by A70C and LI 11C substitutions. Introduction of such a non-native disulfide bond can hold the CD4-BS2 in its functional "trimer-like" conformation in either a monomer of the recombinant gpl20, or in the context of a HIV-1 Env ectodomain trimer including the recombinant gpl20. In some embodiments, introduction of the non- native disulfide bond can hold a tryptophan residues at gpl20 position 69 (HXB2 numbering) inside a cavity occupied by Trpl 12 and Trp427, to maintain the CD4-BS2 in the prefusion mature closed conformation. The recombinant gpl20 comprising the non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109- 113 or a HIV-1 Env ectodomain trimer including the recombinant gpl20 produces an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113.

As disclosed in Example 1, mutation of tyrosine 61 (HXB2 numbering) greatly enhances CD4 binding and consequently HIV-1 infectivity. It is believed that tyrosine 61 interferes with formation of the CD4-BS2 in the prefusion mature closed conformation. Accordingly, in some embodiments, the recombinant gpl20 or a HIV-1 Env ectodomain trimer including the recombinant gpl20 can include a Y61A or a Y61F substitution, which reduced the interference on formation of the CD4-BS2 in the prefusion mature closed conformation when a tyrosine is present at this position. The recombinant gpl20 comprising the Y61A or a Y61F substitution or a HIV-1 Env ectodomain trimer including the recombinant gpl20 produces an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the Y61A or a Y61F substitution.

Additionally, there are at least 2 major glycans (N262 and N302) that partially shield the surface exposure of CD4-BS2 resides. It is believed that targeted elimination of these glycan sites can allow increased exposure of the CD4-BS2 to the immune system. Accordingly, in several embodiments, the recombinant gpl20 or the HIV-1 Env ectodomain trimer including the recombinant gpl20 can include one or more amino acid substitutions to remove the N-linked glycan sequon at position N262 and/or N301. In some embodiments, the recombinant gpl20 or the HIV-1 Env ectodomain trimer including the recombinant gpl20 can include a N262Q substitution and/or a S264A substitution to remove the N-linked glycan sequon at position N262. In additional embodiments, the recombinant gpl20 or the HIV-1 Env ectodomain trimer including the recombinant gpl20 can include a N301Q substitution and/or T303A substitution to remove the N-linked glycan sequon at position N301. The recombinant gpl20 comprising the one or more amino acid substitutions to remove the N-linked glycan sequon at position N262 and/or N301 or a HIV-1 Env ectodomain trimer including the recombinant gpl20 produces an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the one or more amino acid substitutions to remove the N-linked glycan sequon at position N262 and/or N301.

In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 includes one or more non-natural disulfide bonds that stabilize the CD4-BS2 and/or the HIV-1 Env ectodomain trimer in a particular conformation, such as the prefusion mature closed conformation. The prefusion mature closed conformation of the NIV-1 Env trimer has been disclosed, for example, in Pancera et al, Nature, 514, 455-461, 2014 and PCT App. No. PCT/US2015/048729, each of which is incorporated by reference herein in its entirety. In some embodiments, the recombinant gpl20 and or the HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can further include one of more modifications as disclosed in PCT App. No. PCT/US2015/048729 to stabilize the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 in the prefusion mature closed conformation. For example, the HIV-1 Env ectodomain trimer can include a prefusion mature closed conformation wherein the VI V2 domain of each Env ectodomain protomer in the trimer comes together at the membrane distal apex. At the membrane proximal aspect, the HIV-1 Env ectodomain trimer in the prefusion mature closed conformation includes distinct cc6 and cc7 helices; the cc7 helix does not start until after residue 570. For example, in the prefusion mature closed conformation, the interprotomer distance between residues 200 and 313 can be less than 5 Angstroms.

In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can further include a non-natural disulfide bond between gpl20 positions 201 and 433. For example, the non-natural disulfide bond can be introduced by including cysteine substitutions at positions 201 and 433 (e.g., I201C and A433C substitutions). The presence of the non- natural disulfide bond between residues 201 and 433 contributes to the stabilization of the HIV-1 Env ectodomain in the prefusion mature closed conformation. In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can further include a non-natural disulfide bond between gpl20 positions 201 and 433 (e.g., by introduction of 1201 C and A433C substitutions) and can further included the SOSIP mutations, as disclosed below.

In some embodiments, any of the HIV-1 Env ectodomain trimers including the recombinant gpl20 disclosed herein can further include a non-natural disulfide bond between cysteine substitutions at one of gpl20 positions 71-75 and one of gp41 positions 553-559. For example, the non-natural disulfide bond can be introduced by including cysteine substitutions at gpl20 position 73 and gp41 position 557 (e.g., A7C and R557C substitutions). The presence of the non-natural disulfide bond between residues 73 and 557 contributes to the stabilization of the HIV-1 Env ectodomain in the prefusion mature closed conformation. In some embodiments, the disclosed recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can further include a non-natural disulfide bond between gpl20 positions 73 and 557 (e.g., by introduction of A73C and R557C substitutions) and can further included the SOSIP mutations, and/or a non-natural disulfide bond between gpl20 positions 201 and 433. In some embodiments, the distance between residues 73 and 557 may be reduced by introduction of spacer residues, such as glycines, on both sides of position 73 or 557.

In some embodiments, an HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can further include the "SOSIP" substitutions, which include a non-natural disulfide bond between cysteine residues introduced at HIV- 1 Env positions 501 and 605 (for example, by A501C and T605C substitutions), and a proline residue introduced at HIV- 1 Env positions 559 (for example, by an I559P substitution). The presence of the non-natural disulfide bond between positions 501 and 605 and the proline residue at position 559 contributes to the stabilization of the HIV-1 Env ectodomain in the prefusion mature closed conformation.

In some embodiments, the disclosed recombinant gpl20 and/or an HIV-1 Env ectodomain trimer including the recombinant gpl20 can further include an N-linked glycosylation site at gpl20 position 332 (if not already present on the ectodomain). For example, by T332N substitution in the case of BG505 based immunogens. The presence of the glycosylation site at N332 allows for binding by 2G12 antibody.

In some embodiments, the disclosed recombinant gpl20 and/or an HIV-1 Env ectodomain trimer including the recombinant gpl20 can further include a lysine substitution at gpl20 position 168 (if not already present on the ectodomain). For example, the lysine residue can be added by amino acid substitution (such as an E168K substitution in the case of the JR-FL based immunogens). The presence of the lysine residue at position 168 allows for binding of particular broadly neutralizing antibodies to the VI V2 loop of gpl20.

In some embodiments, the disclosed recombinant gpl20 and/or an HIV-1 Env ectodomain trimer including the recombinant gpl20 can further include an arginine substitution at gpl20 position 368 (if not already present on the ectodomain). For example, the arginine residue can be added by amino acid substitution (such as a D368R substitution). The presence of the arginine residue at position 368 reduces binding of CD4 to the HIV-1 Env ectodomain to inhibit the trimer from adopting the CD4-bound conformation.

In some embodiments, the HIV-1 Env ectodomain trimers including the recombinant gpl20 disclosed herein can include mutations to add an N- linked glycan sequon at position 504, position 661, or positions 504 and 661, to increase glycosylation of the membrane proximal region of the ectodomain.

Native HIV- 1 Env sequences include a furin cleavage site between positions 508 and 512 (HXB2 numbering), that separates gpl20 and gp41. Any of the disclosed recombinant HIV-1 Env ectodomains can further include an enhanced cleavage site between gpl20 and gp41 proteins. The enhanced cleavage cite can include, for example, substitution of six arginine resides for the four residues of the native cleavage site (e.g., REKR (SEQ ID NO: 10) to RRRRRR (SEQ ID NO: 11). It will be understood that protease cleavage of the furin or enhanced cleavage site separating gpl20 and gp41 can remove a few amino acids from either end of the cleavage site.

The purified form of the recombinant gpl20 typically does not include a signal peptide (for example, the gpl20 typically does not include gpl20 residues 1-30), as the signal peptide is proteolytically cleaved during cellular processing. In some embodiments, the n-terminal residue of the gpl20 is one of HIV-1 Env positions 1-35, and the c-terminal residue of the recombinant gpl20 is one of HIV- 1 Env positions 503-511. In some embodiments, the n-terminal residue of the recombinant gpl20 is HIV-1 Env position 31 and the c-terminal residue of the recombinant gpl20 is HIV-1 Env position 511.

The disclosed HIV-1 Env ectodomain trimers include the recombinant gpl20 and a gp41 ectodomain. The recombinant gpl20 typically does not include a signal peptide (for example, the recombinant gpl20 typically does not include gpl20 residues 1-30), as the signal peptide is proteolytically cleaved during cellular processing. Additionally, the gp41 ectodomain includes the extracellular portion of gp41 (e.g., positions 512-664). In embodiments including a soluble recombinant HIV-1 Env ectodomain, the gp41 ectodomain is not linked to a transmembrane domain or other membrane anchor. However, in embodiments including a membrane anchored recombinant HIV-1 Env ectodomain the gp41 ectodomain can be linked to a transmembrane domain (such as, but not limited to, an HIV-1 Env transmembrane domain).

In some embodiments, the HIV-1 Env ectodomain trimer includes the recombinant gpl20 and a gp41 ectodomain and the N-terminal residue of the gpl20 is one of HIV-1 Env positions 1-35;

the C-terminal residue of the gpl20 is one of HIV- 1 Env positions 503-511 ;

the N-terminal residue of the gp41 ectodomain is one of HIV-1 Env positions 512-522; and/or the C-terminal residue of the gp41 ectodomain is one of HIV-1 Env positions 624-705.

In one non-limiting example, the HIV-1 Env ectodomain trimer includes the recombinant gpl20 and the gp41 ectodomain, wherein the n-terminal residue of the recombinant gpl20 is HIV-1 Env position 31; the c-terminal residue of the recombinant gpl20 is HIV-1 Env position 511; the n-terminal residue of the gp41 ectodomain is HIV-1 Env position 512; and the c-terminal residue of the gp41 ectodomain is HIV-1 Env position 664. In some embodiments, the C-terminal residue of the recombinant HIV-1 Env ectodomain is position 683 (the entire ectodomain, terminating just before the transmembrane domain). In additional embodiments, the C-terminal residue of the recombinant HIV-1 Env ectodomain is position 707 (the entire ectodomain, terminating just after the transmembrane domain).

HIV-1 can be classified into four groups: the "major" group M, the "outlier" group O, group N, and group P. Within group M, there are several genetically distinct clades (or subtypes) of HIV-1. The disclosed recombinant HIV-1 Env proteins can be derived from any type of HIV, such as groups M, N, O, or P, or clade, such as clade A, B, C, D, F, G, H, J, or K, and the like. HIV-1 Env proteins from the different HIV-1 clades, as well as nucleic acid sequences encoding such proteins and methods for the manipulation and insertion of such nucleic acid sequences into vectors, are known (see, e.g., HIV Sequence Compendium, Division of AIDS, National Institute of Allergy and Infectious Diseases (2013); HIV Sequence Database (hiv-web.lanl.gov/content/hiv-db/mainpage.html); see, e.g., Sambrook et al. (Molecular Cloning: A

Laboratory Manual, 4^th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Exemplary native HIV-1 Env protein sequences are available in the HIV Sequence Database (hiv-web.lanl.gov/content/hiv- db/mainpage.html) .

In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimers including the recombinant gpl20 disclosed herein can include an amino acid sequence of a native gpl20 or HIV- 1 Env protein, for example, from genetic subtype A-F as available in the HIV Sequence Database (hiv- web. lanl.gov/content/hiv-db/mainpage.html) or as set forth in Table 1, or an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) identical thereto, that has been modified to include the one or more amino acid substitutions as discussed above, for example, to stabilize the CD4-BS2 and/or the HIV-1 ectodomain trimer in the prefusion mature closed conformation.

Table 1. HIV-1 Env sequences

CTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFG NNKTI IFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDT ITLPCRIKQI INMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGG GDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQREKRAVGIGALFLGFLGAAGSTMG AASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ QLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEES QNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRV RQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFS YHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGT DRVIEWQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 1)

BG505 A RVMGIQRNCQHLFRWGTMILGMI I ICSAAENLWVTVYYGVPVWKDAETTLFCASDAKA

YETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI ISLWDQSLKPC VKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDWQINE NQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTG PCPSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINC TRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCTVSKATWNETLGKWKQLRKHFGNN TIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITL PCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDM RDNWRSELYKYKWKIEPLGVAPTRAKRRWGREKRAVGIGAVFLGFLGAAGSTMGAAS TLTVQARNLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLL GIWGCSGKLICTTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQI IYGLLEESQNQ QEKNEQDLLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVIHRVRQG YSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTRLVSGFLALAWDDLRSLCLFCYHR LRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAYWGRELKISAINLFDTIAIAV AEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL (SEQ ID NO: 2)

CONSENS A MRVMGIQRNCQHLLRWGTMILGMI I ICSAAENLWVTVYYGVPVWKDAETTLFCASDAKA US_A1 YETEMHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDI ISLWDQSLKPC

VKLTPLCVTLNCSNVNVTNNTTNTHEEEIKNCSFNMTTELRDKKQKVYSLFYRLDWQI NENNSNSSYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKEFNGTGPCKN VSTVQCTHGIKPWSTQLLLNGSLAEEEVI IRSENITNNAKTI IVQLTKPVKINCTRPN NNTRKSIRIGPGQAFYATGDI IGDIRQAHCNVSRSEWNKTLQKVAKQLRKYFKNKTI IF TNSSGGDLEITTHSFNCGGEFFYCNTSGLFNSTWNNGTMKNTITLPCRIKQI INMWQRA GQAMYAPPIQGVIRCESNITGLLLTRDGGNNNTNETFRPGGGDMRDNWRSELYKYKWK IEPLGVAPTRAKRRWEREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIV QQQSNLLRAIEAQQHLLKLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTNV PWNSSWSNKSQNEIWDNMTWLQWDKEISNYTHIIYNLIEESQNQQEKNEQDLLALDKWA NLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSVINRVRQGYSPLSFQTHTPNPRG LDRPGRIEEEGGEQGRDRSIRLVSGFLALAWDDLRSLCLFSYHRLRDFILIAARTVELL GHSSLKGLRLGWEGLKYLWNLLLYWGRELKISAINLVDTIAIAVAGWTDRVIEIGQRIG RAILHIPRRIRQGLERALL (SEQ ID NO: 3)

CONSENSU A RVMGTQRNYQHLWRWGILILGMLIMCKATDLWVTVYYGVPVWKDADTTLFCASDAKAY

S_A2 DTEVHNVWATHACVPTDPNPQEVNLENVTEDFNMWKNNMVEQMHEDIISLWDQSLKPCV

KLTPLCVTLNCSNANTTNNSTMEEIKNCSYNITTELRDKTQKVYSLFYKLDWQLDESN KSEYYYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDPRFNGTGSCNNVSS VQCTHGIKPVASTQLLLNGSLAEGKVMIRSENITNNAKNIIVQFNKPVPITCIRPNNNT RKSIRFGPGQAFYTNDI IGDIRQAHCNINKTKWNATLQKVAEQLREHFPNKTIIFTNSS GGDLEITTHSFNCGGEFFYCNTTGLFNSTWKNGTTNNTEQMITLPCRIKQI INMWQRVG RAMYAPPIAGVIKCTSNITGI ILTRDGGNNETETFRPGGGDMRDNWRSELYKYKWKIE PLGVAPTRAKRRWEREKRAVGMGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQ QSNLLKAIEAQQHLLKLTVWGIKQLQARVLALERYLQDQQLLGIWGCSGKLICATTVPW NSSWSNKTQEEIWNNMTWLQWDKEISNYTNI IYKLLEESQNQQEKNEQDLLALDKWANL WNWFNITNWLWYIRIFIMIVGGLIGLRIVIAI ISWNRVRQGYSPLSFQIPTPNPEGLD RPGRIEEGGGEQGRDRSIRLVSGFLALAWDDLRSLCLFSYHRLRDCILIAARTVELLGH SSLKGLRLGWEGLKYLWNLLLYWGRELKNSAISLLDTIAVAVAEWTDRVIEIGQRACRA ILNIPRRIRQGFERALL (SEQ ID NO: 4)

CONSENSU B RVKGIRKNYQHLWRWGTMLLGMLMICSAAEKLWVTVYYGVPVWKEATTTLFCASDAKA S_B YDTEVHNVWATHACVPTDPNPQEWLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPC VKLTPLCVTLNCTDLMNATNTNTTI IYRWRGEIKNCSFNITTSIRDKVQKEYALFYKLD WPIDNDNTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPC TNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSENFTDNAKTIIVQLNESVEINCTR PNNNTRKSIHIGPGRAFYTTGEI IGDIRQAHCNISRAKWNNTLKQIVKKLREQFGNKTI VFNQSSGGDPEIVMHSFNCGGEFFYCNTTQLFNSTWNGTWNNTEGNITLPCRIKQI INM WQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNETEIFRPGGGDMRDNWRSELYKYK WKIEPLGVAPTKAKRRWQREKRAVGIGAMFLGFLGAAGSTMGAASMTLTVQARQLLS GIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICT TAVPWNASWSNKSLDEIWDNMTWMEWEREIDNYTSLIYTLIEESQNQQEKNEQELLELD KWASLWNWFDITNWLWYIKIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTRLPA PRGPDRPEGIEEEGGERDRDRSGRLVDGFLALIWDDLRSLCLFSYHRLRDLLLIVTRIV ELLGRRGWEVLKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEWQRACRAIL HIPRRIRQGLERALL (SEQ ID NO: 5)

CONSENSU C RVRGILRNCQQWWIWGILGFWMLMICNWGNLWVTVYYGVPVWKEAKTTLFCASDAKA

s_c YEKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDI ISLWDQSLKPC

VKLTPLCVTLNCTNATNATNTMGEIKNCSFNITTELRDKKQKVYALFYRLDIVPLNENN SYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCT HGIKPWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPNNNTRKSI RIGPGQTFYATGDI IGDIRQAHCNISEDKWNKTLQKVSKKLKEHFPNKTIKFEPSSGGD LEITTHSFNCRGEFFYCNTSKLFNSTYNSTNSTITLPCRIKQI INMWQEVGRAMYAPPI AGNITCKSNITGLLLTRDGGKNNTETFRPGGGDMRDNWRSELYKYKWEIKPLGIAPTK AKRRWEREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAI EAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKLICTTAVPWNSSWSNKS QEDIWDNMTWMQWDREISNYTDTIYRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDITN WLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLTPNPRGPDRLGRIEEE GGEQDRDRSIRLVSGFLALAWDDLRSLCLFSYHRLRDFILIAARAVELLGRSSLRGLQR GWEALKYLGSLVQYWGLELKKSAISLLDTIAIAVAEGTDRI IELIQRICRAIRNIPRRI RQGFEAALQ (SEQ ID NO: 6)

CONSENSU D RVRGIQRNYQHLWRWGIMLLGMLMICSVAENLWVTVYYGVPVWKEATTTLFCASDAKS

S_D YKTEAHNIWATHACVPTDPNPQEIELENVTENFNMWKNNMVEQMHEDI ISLWDQSLKPC

VKLTPLCVTLNCTDVKRNNTSNDTNEGEMKNCSFNITTEIRDKKKQVHALFYKLDWPI DDNNSNTSYRLINCNTSAITQACPKVTFEPIPIHYCAPAGFAILKCKDKKFNGTGPCKN VSTVQCTHGIRPWSTQLLLNGSLAEEE11 IRSENLTNNAKI I IVQLNESVTINCTRPY NNTRQRTPIGPGQALYTTRIKGDIRQAHCNISRAEWNKTLQQVAKKLGDLLNKTTI IFK PSSGGDPEITTHSFNCGGEFFYCNTSRLFNSTWNNTKWNSTGKITLPCRIKQIINMWQG VGKAMYAPPIEGLIKCSSNITGLLLTRDGGANNSHNETFRPGGGDMRDNWRSELYKYKV VKIEPLGVAPTRAKRRWEREKRAIGLGAMFLGFLGAAGSTMGAASMTLTVQARQLLSG IVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKHICTT TVPWNSSWSNKSLDEIWNNMTWMEWEREIDNYTGLIYSLIEESQNQQEKNEQELLELDK WASLWNWFSITQWLWYIKIFIMIVGGLIGLRIVFAVLSLVNRVRQGYSPLSFQTLLPAP RGPDRPEGIEEEGGEQGRGRSIRLVNGFSALIWDDLRNLCLFSYHRLRDLILIAARIVE LLGRRGWEALKYLWNLLQYWIQELKNSAISLFDTTAIAVAEGTDRVIEIVQRACRAILN IPTRIRQGLERALL (SEQ ID NO: 7)

CONSENSU F RVRGMQRNWQHLGKWGLLFLGILI ICNAAENLWVTVYYGVPVWKEATTTLFCASDAKS

S_F1 YEKEVHNVWATHACVPTDPNPQEWLENVTENFDMWKNNMVEQMHTDI ISLWDQSLKPC

VKLTPLCVTLNCTDVNATNNDTNDNKTGAIQNCSFNMTTEVRDKKLKVHALFYKLDIVP ISNNNSKYRLINCNTSTITQACPKVSWDPIPIHYCAPAGYAILKCNDKRFNGTGPCKNV STVQCTHGIKPWSTQLLLNGSLAEEDIIIRSQNISDNAKTIIVHLNESVQINCTRPNN NTRKSIHLGPGQAFYATGEIIGDIRKAHCNISGTQWNKTLEQVKAKLKSHFPNKTIKFN SSSGGDLEITMHSFNCRGEFFYCNTSGLFNDTGSNGTITLPCRIKQIVNMWQEVGRAMY AAPIAGNITCNSNITGLLLTRDGGQNNTETFRPGGGNMKDNWRSELYKYKWEIEPLGV APTKAKRQWKRERRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQNNL LRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGLWGCSGKLICTTNVPWNSSW SNKSQDEIWNNMTWMEWEKEISNYSNIIYRLIEESQNQQEKNEQELLALDKWASLWNWF DISNWLWYIKIFIMIVGGLIGLRIVFAVLSIVNRVRKGYSPLSLQTLIPSPREPDRPEG IEEGGGEQGKDRSVRLVNGFLALVWDDLRNLCLFSYRHLRDFILIAARIVDRGLRRGWE ALKYLGNLTQYWSQELKNSAISLLNTTAIWAEGTDRVIEALQRAGRAVLNIPRRIRQG LERALL (SEQ ID NO: 8)

CONSENSU F RVREMQRNWQHLGKWGLLFLGILI ICNAADNLWVTVYYGVPVWKEATTTLFCASDAKA

S_F2 YEREVHNVWATYACVPTDPSPQELVLGNVTENFNMWKNNMVDQMHEDI ISLWDQSLKPC

VKLTPLCVTLNCTDVNVTINTTNVTLGEIKNCSFNITTEIKDKKKKEYALFYRLDWPI NNSIVYRLISCNTSTVTQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGLCRNVST VQCTHGIRPWSTQLLLNGSLAEEDIIIRSENISDNTKTIIVQFNRSVEINCTRPNNNT RKSIRIGPGRAFYATGDI IGDIRKAYCNINRTLWNETLKKVAEEFKNHFNITVTFNPSS GGDLEITTHSFNCRGEFFYCNTSDLFNNTEVNNTKTITLPCRIRQFVNMWQRVGRAMYA PPIAGQIQCNSNITGLLLTRDGGKNGSETLRPGGGDMRDNWRSELYKYKWKIEPLGVA PTKAKRQWQREKRAVGIGAVLLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLL KAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTNVPWNSSWS NKSQDEIWDNMTWMQWEKEISNYTDTIYRLIEDAQNQQEKNEQDLLALDKWDNLWSWFT ITNWLWYIKIFIMIVGGLIGLRIVFAVLSWNRVRQGYSPLSLQTLIPNPRGPERPGGI EEEGGEQDRDRSIRLVSGFLALAWDDLRSLCLFSYRHLRDFILIAARTVDMGLKRGWEA LKYLWNLPQYWGQELKNSAISLLDTTAIAVAEGTDRI IEVLQRAGRAVLHIPRRIRQGF ERALL (SEQ ID NO: 9)

In view of the conservation and breadth of knowledge of HIV- 1 Env sequences, the person of ordinary skill in the art can easily identify corresponding HIV-1 Env amino acid positions between different HrV-1 Env strains and subtypes. The HXB2 numbering system has been developed to assist comparison between different HIV- 1 amino acid and nucleic acid sequences. The person of ordinary skill in the art is familiar with the HXB2 numbering system (see, e.g., Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken CL, Foley B, Hahn B, McCutchan F, Mellors JW, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety). The numbering of amino acid substitutions disclosed herein is made according to the HXB2 numbering system, unless context indicates otherwise.

It is understood in the art that some variations can be made in the amino acid sequence of a protein without affecting the activity of the protein. Such variations include insertion of amino acid residues, deletions of amino acid residues, and substitutions of amino acid residues. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering technique known to those skilled in the art. Examples of such techniques are found in see, e.g., Sambrook et al.

(Molecular Cloning: A Laboratory Manual, 4^th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013, both of which are incorporated herein by reference in their entirety.

In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 is soluble in aqueous solution (for example, the HIV-1 Env ectodomain trimer does not include the gp41 TM or CT domains). In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 dissolves to a concentration of at least 0.5 mg/ml (such as at least 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml or at least 5.0 mg/ml) in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22 degrees Celsius) and remains dissolved for at least for at least 12 hours (such as at least 24 hours, at least 48 hours, at least one week, at least two weeks, or more time). In one embodiment, the phosphate buffered saline includes NaCl (137 mM), KC1 (2.7 mM), Na2HP04 (10 mM), KH2PO4 (1.8 mM) at pH 7.4. In some embodiments, the phosphate buffered saline further includes CaC (1 mM) and MgCk (0.5 mM). The person of skill in the art is familiar with methods of determining if a protein remains in solution over time. For example, the concentration of the protein dissolved in an aqueous solution can be tested over time using standard methods.

The recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 can be derivatized or linked to another molecule (such as another peptide or protein). In general, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 is derivatized such that the binding to broadly neutralizing antibodies to a trimer of the recombinant HIV-1 ectodomain, such as PGT122, is not affected adversely by the derivatization or labeling. For example, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as an antibody or protein or detection tag.

In some embodiments, the recombinant gpl20 and/or the HIV-1 Env ectodomain trimer including the recombinant gpl20 includes one or more non-natural disulfide bonds that stabilize the CD4-BS2 and/or the HIV-1 Env ectodomain trimer in the prefusion mature closed conformation. The cysteine residues that form the disulfide bond can be introduced into a native HIV- 1 sequence by one or more amino acid substitutions. For example, the amino acid positions of the cysteines are typically within a sufficiently close distance for formation of a disulfide bond in the prefusion mature closed conformation of the HIV- 1 Env protein trimer. Methods of using three-dimensional structure data to determine if two residues are within a sufficiently close distance to one another for disulfide bond formation are known (see, e.g., Peterson et al., Protein engineering, 12:535-548, 1999 and Dombkowski, Bioinformatics, 19: 1852-1853, 3002 (disclosing DISULFIDE BY DESIGN™), each of which is incorporated by reference herein). Residues can be selected manually, based on the three dimensional structure of the HIV-1 Env trimer in a prefusion mature closed conformation provided herein, or a software, such as DISULFIDEBYDESIGN™, can be used. Without being bound by theory, ideal distances for formation of a disulfide bond are generally considered to be about -5.6A for Ca-Ca distance, -2.02 A for Sy-Sy distance, and 3.5-4.25 A for Οβ-Οβ distance (using the optimal rotomer). The person of ordinary skill in the art will appreciate that variations from these distances are included when selecting residues in a three dimensional structure that can be substituted for cysteines for introduction of a disulfide bond. For example, in some embodiments the selected residues have a Ca-Ca distance of less than 7.0 A and/or a C -C distance of less than 4.7 A. In some embodiments the selected residues have a Ca-Ca distance of from 2.0-8.0 A and/or a C -C distance of from 2.0-5.5 A. In some embodiments, the amino acid positions of the cysteines are within a sufficiently close distance for formation of a disulfide bond in the prefusion mature closed conformation, but not the CD4-bound open conformation of the HIV-1 Env protein. Single Chain HIV-1 Env proteins

In some embodiments, the HIV-1 Env ectodomain trimer including the recombinant gpl20 can be made of three single chain HIV-1 Env ectodomains, which each include a single polypeptide chain including the gpl20 polypeptide and the gp41 ectodomain. Native HIV-1 Env sequences include a furin cleavage site at position 511 (e.g., REKR5_{1 1}), which is cleaved by a cellular protease to generate the gpl20 and gp41 polypeptides. The single chain proteins do not include the furin cleavage site separating the gpl20 and gp41 polypeptides; therefore, when produced in cells, the Env polypeptide is not cleaved into separate gpl20 and gp41 polypeptides.

Single chain HIV-1 Env ectodomains can be generated by mutating the furin cleavage site to prevent cleave and formation of separate gpl20 and gp41 polypeptide chains. In some embodiments, the gpl20 and gp41 polypeptides in the single chain HIV-1 Env ectodomains are joined by a linker, such as a peptide linker. Examples of peptide linkers that can be used include glycine, serine, and glycine-serine linkers. In some embodiments, the peptide liker can comprise a 10 amino acid glycine- serine peptide linker, such as a peptide linker comprising the amino acid sequence set forth as SEQ ID NOs: 12 (GGSGGGGSGG). In some embodiments, the single chain HIV-1 Env ectodomains can include a heterologous peptide linker between one of HIV-1 Env residues 507 and 512, 503 and 519, 504 and 519, 503 and 522, or 504 and 522. In some embodiments, the HIV-1 Env ectodomain trimer including the recombinant gpl20 as disclosed herein can include three single chain HIV-1 Env ectodomains each comprising a heterologous peptide linker (such as a 10 amino acid glycine serine linker) between HIV-1 Env residues 507 and 512.

Any of the stabilizing mutations (or combinations thereof) disclosed herein can be included in the single chain HIV-1 Env ectodomain as long as the single chain HIV-1 Env ectodomain retains the desired properties (e.g., the HIV-1 Env prefusion mature closed conformation).

Membrane anchored embodiments

In some embodiments, the HIV-1 Env ectodomain trimer including the recombinant gpl20 can be a membrane anchored HIV-1 Env ectodomain trimer, for example, the HIV-1 Env ectodomains in the trimer can each be linked to a transmembrane domain. The transmembrane domain can be linked to any portion of the HIV-1 Env ectodomain, as long as the presence of the transmembrane domain does not disrupt the structure of the HIV-1 Env ectodomain, or its ability to elicit an immune response to HIV-1. In non-limiting examples, the transmembrane domain can be linked to the N- or C-terminal residue of a gpl20 polypeptide, or the C-terminal residue of a gp41 ectodomain included in the HIV-1 Env ectodomain. One or more peptide linkers (such as a gly-ser linker, for example, a 10 amino acid glycine-serine peptide linker, such as a peptide linker comprising the amino acid sequence set forth as SEQ ID NO: 12 (GGSGGGGSGG) can be used to link the transmembrane domain and the gpl20 or gp41 protein. In some embodiments a native HIV- 1 Env MPER sequence can be used to link the transmembrane domain and the gpl20 or gp41 protein. In some embodiments, the HIV-1 Env ectodomain trimer including the recombinant gpl20 can include a full HIV-1 Env transmembrane and cytosolic regions. Non-limiting examples of transmembrane domains for use with the disclosed embodiments include the BG505 TM domain (KIFIMIVGGLIGLRIVFAVLSVIHRVR, SEQ ID NO: 13), the Influenza A Hemagglutinin TM domain (ILAIYSTVASSLVLLVSLGAISF, SEQ ID NO: 14), and the Influenza A Neuraminidase TM domain (IITIGSICMVVGIISLILQIGNIISIWVS, SEQ ID NO: 15).

The recombinant HIV-1 Env ectodomain linked to the transmembrane domain can include any of the stabilizing mutations provided herein (or combinations thereof) as long as the recombinant HIV-1 Env ectodomain linked to the transmembrane domain retains the desired properties (e.g., the HIV-1 Env prefusion mature closed conformation). Linkage to a Trimerization Domain

In some embodiments, the HIV-1 Env ectodomain trimer including the recombinant gpl20 can be linked to a trimerization domain, for example, the C -terminus of the gp41 protein included in the HIV-1 Env ectodomain can be linked to the trimerization domain. The trimerization domain can promotes trimerization of the three protomers of the recombinant HIV- 1 Env protein. Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include: the GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407), the trimerization motif from the lung surfactant protein (Hoppe et al. 1994 FEBS Lett 344: 191-195), collagen (McAlinden et al. 2003 J Biol Chem

278:42200-42207), and the phage T4 fibritin Foldon (Miroshnikov et al. 1998 Protein Eng 11 :329-414), any of which can be linked to the recombinant HIV- 1 Env ectodomain (e.g., by linkage to the C-terminus of the gp41 polypeptide to promote trimerization of the recombinant HIV-1 protein, as long as the recombinant

HIV-1 Env ectodomain retains specific binding activity for a mature closed conformation specific antibody, prefusion- specific antibody (e.g., PGT122), and/or includes a HIV-1 Env mature closed conformation.

In some examples, the recombinant HIV-1 Env ectodomain can be linked to a T4 fibritin Foldon domain, for example, the recombinant HIV-1 Env ectodomain can include a gp41 polypeptide with a Foldon domain linked to its C-terminus. In specific examples, the T4 fibritin Foldon domain can include the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTF (SEQ ID NO: 16), which adopts a β-propeller conformation, and can fold and trimerize in an autonomous way (Tao et al. 1997 Structure 5:789-798).

Typically, the heterologous trimerization domain is positioned C-terminal to the gp41 protein. Optionally, the heterologous trimerization is connected to the recombinant HIV-1 Env ectodomain via a linker, such as an amino acid linker. Exemplary linkers include Gly or Gly-Ser linkers, such as SEQ ID NO: 12 (GGSGGGGSGG). Some embodiments include a protease cleavage site for removing the trimerization domain from the HIV- 1 polypeptide, such as, but not limited to, a thrombin site between the recombinant HIV-1 Env ectodomain and the trimerization domain.

Exemplary sequences

The following table provides sequences of HIV-1 Env proteins including A70C and LI 11C substitutions. The recombinant gpl20 or HIV-1 Env ectodomain trimer including the recombinant gpl20 disclosed herein can include relevant sequences of the recombinant HIV-1 Env sequences provided below.

Any of the other stabilizing mutations provided herein can also be included in the recombinant gpl20 or HIV-1 Env ectodomain trimer.

Name Clade Env sequence

HXB2 A70C- B MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYD L111C TEVHNVWCTHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDI ISCWDQSLKPCVKLTP

LCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDN DTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCT HGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTI IVQLNTSVEINCTRPNNNTRKRIRIQ RGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIV THSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAM YAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGV APTKAKRRWQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRA IEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLE QIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYI KLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDR SIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQE LKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL (SEQ ID NO: 21)

BG505 A70C- A MRVMGIQRNCQHLFRWGTMILGMI IICSAAENLWVTVYYGVPVWKDAETTLFCASDAKAYET L111C EKHNVWCTHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII SCWDQSLKPCVKLTPL

CVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNS NKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTH GIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGP GQAFYATGDI IGDIRQAHCTVSKATWNETLGKWKQLRKHFGNNTI IRFANSSGGDLEVTTH SFNCGGEFFYCNTSGLFNSTWI SNTSVQGSNSTGSNDSITLPCRIKQI INMWQRIGQAMYAP PIQGVIRCVSNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPT RAKRRVVGREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAIEA QQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNRNLSEIW DNMTWLQWDKEI SNYTQI IYGLLEESQNQQEKNEQDLLALDKWASLWNWFDI SNWLWYIKIF IMIVGGLIGLRIVFAVLSVIHRVRQGYSPLSFQTHTPNPRGLDRPERIEEEDGEQDRGRSTR LVSGFLALAWDDLRSLCLFCYHRLRDFILIAARIVELLGHSSLKGLRLGWEGLKYLWNLLAY WGRELKISAINLFDTIAIAVAEWTDRVIEIGQRLCRAFLHIPRRIRQGLERALL (SEQ ID NO: 22)

CONSENSUS. A MRVMGIQRNCQHLLRWGTMILGMI IICSAAENLWVTVYYGVPVWKDAETTLFCASDAKAYET Al A70C- EMHNVWCTHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII SCWDQSLKPCVKLTPL L111C CVTLNCSNVNVTNNTTNTHEEEIKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENNSNSSY

RLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKEFNGTGPCKNVSTVQCTHGIKP WSTQLLLNGSLAEEEVIIRSENITNNAKTIIVQLTKPVKINCTRPNNNTRKSIRIGPGQAF YATGDI IGDIRQAHCNVSRSEWNKTLQKVAKQLRKYFKNKTI IFTNSSGGDLEITTHSFNCG GEFFYCNTSGLFNSTWNNGTMKNTITLPCRIKQI INMWQRAGQAMYAPPIQGVIRCESNITG LLLTRDGGNNNTNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRWEREKRAVG IGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLKLTVWGIKQL QARVLAVERYLKDQQLLGIWGCSGKLICTTNVPWNSSWSNKSQNEIWDNMTWLQWDKEISNY THIIYNLIEESQNQQEKNEQDLLALDKWANLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFA VLSVINRVRQGYSPLSFQTHTPNPRGLDRPGRIEEEGGEQGRDRSIRLVSGFLALAWDDLRS LCLFSYHRLRDFILIAARTVELLGHSSLKGLRLGWEGLKYLWNLLLYWGRELKI SAINLVDT IAIAVAGWTDRVIEIGQRIGRAILHIPRRIRQGLERALL (SEQ ID NO: 23)

CONSENSUS. A MRVMGTQRNYQHLWRWGILILGMLIMCKATDLWVTVYYGVPVWKDADTTLFCASDAKAYDTE A2 A70C- VHNVWCTHACVPTDPNPQEVNLENVTEDFNMWKNNMVEQMHEDI ISCWDQSLKPCVKLTPLC L111C VTLNCSNANTTNNSTMEEIKNCSYNITTELRDKTQKVYSLFYKLDVVQLDESNKSEYYYRLI

NCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDPRFNGTGSCNNVSSVQCTHGIKPVAS TQLLLNGSLAEGKVMIRSENITNNAKNI IVQFNKPVPITCIRPNNNTRKSIRFGPGQAFYTN DI IGDIRQAHCNINKTKWNATLQKVAEQLREHFPNKTI IFTNSSGGDLEITTHSFNCGGEFF YCNTTGLFNSTWKNGTTNNTEQMITLPCRIKQIINMWQRVGRAMYAPPIAGVIKCTSNITGI ILTRDGGNNETETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRWEREKRAVGMG AVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLKAIEAQQHLLKLTVWGIKQLQA RVLALERYLQDQQLLGIWGCSGKLICATTVPWNSSWSNKTQEEIWNNMTWLQWDKEISNYTN IIYKLLEESQNQQEKNEQDLLALDKWANLWNWFNITNWLWYIRIFIMIVGGLIGLRIVIAII SVVNRVRQGYSPLSFQIPTPNPEGLDRPGRIEEGGGEQGRDRSIRLVSGFLALAWDDLRSLC LFSYHRLRDCILIAARTVELLGHSSLKGLRLGWEGLKYLWNLLLYWGRELKNSAISLLDTIA VAVAEWTDRVIEIGQRACRAILNIPRRIRQGFERALL (SEQ ID NO: 24)

CONSENSUS. B MRVKGIRKNYQHLWRWGTMLLGMLMICSAAEKLWVTVYYGVPVWKEATTTLFCASDAKAYDT B A70C- EVHNVWCTHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDII SCWDQSLKPCVKLTPL L111C CVTLNCTDLMNATNTNTTIIYRWRGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNDNT

SYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGI RPWSTQLLLNGSLAEEEWIRSENFTDNAKTIIVQLNESVEINCTRPNNNTRKSIHIGPGR AFYTTGEI IGDIRQAHCNISRAKWNNTLKQIVKKLREQFGNKTIVFNQSSGGDPEIVMHSFN CGGEFFYCNTTQLFNSTWNGTWNNTEGNITLPCRIKQI INMWQEVGKAMYAPPIRGQIRCSS NITGLLLTRDGGNNETEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRVVQREKR AVGIGAMFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGI KQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDEIWDNMTWMEWEREI DNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDITNWLWYIKIFIMIVGGLVGLRI VFAVLSIVNRVRQGYSPLSFQTRLPAPRGPDRPEGIEEEGGERDRDRSGRLVDGFLALIWDD LRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEVLKYWWNLLQYWSQELKNSAVSLLNATAIA VAEGTDRVIEWQRACRAILHIPRRIRQGLERALL (SEQ ID NO: 25)

CONSENSUS. C MRVRGILRNCQQWWIWGILGFWMLMICNWGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEK C A70C- EVHNVWCTHACVPTDPNPQEIVLENVTENFNMWKNDMVDQMHEDII SCWDQSLKPCVKLTPL L111C CVTLNCTNATNATNTMGEIKNCSFNITTELRDKKQKVYALFYRLDIVPLNENNSYRLINCNT

SAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLL LNGSLAEEEI IIRSENLTNNAKTI IVHLNESVEIVCTRPNNNTRKSIRIGPGQTFYATGDII GDIRQAHCNI SEDKWNKTLQKVSKKLKEHFPNKTIKFEPSSGGDLEITTHSFNCRGEFFYCN TSKLFNSTYNSTNSTITLPCRIKQIINMWQEVGRAMYAPPIAGNITCKSNITGLLLTRDGGK NNTETFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKAKRRWEREKRAVGIGAVFLGFLG AAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERY LKDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQEDIWDNMTWMQWDREISNYTDTIYRLLED SQNQQEKNEKDLLALDSWKNLWNWFDITNWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQ GYSPLSFQTLTPNPRGPDRLGRIEEEGGEQDRDRSIRLVSGFLALAWDDLRSLCLFSYHRLR DFILIAARAVELLGRSSLRGLQRGWEALKYLGSLVQYWGLELKKSAISLLDTIAIAVAEGTD RIIELIQRICRAIRNIPRRIRQGFEAALQ (SEQ ID NO: 26)

CONSENSUS. D MRVRGIQRNYQHLWRWGIMLLGMLMICSVAENLWVTVYYGVPVWKEATTTLFCASDAKSYKT D A70C- EAHNIWCTHACVPTDPNPQEIELENVTENFNMWKNNMVEQMHEDII SCWDQSLKPCVKLTPL L111C CVTLNCTDVKRNNTSNDTNEGEMKNCSFNITTEIRDKKKQVHALFYKLDVVPIDDNNSNTSY

RLINCNTSAITQACPKVTFEPIPIHYCAPAGFAILKCKDKKFNGTGPCKNVSTVQCTHGIRP WSTQLLLNGSLAEEEII IRSENLTNNAKI IIVQLNESVTINCTRPYNNTRQRTPIGPGQAL YTTRIKGDIRQAHCNI SRAEWNKTLQQVAKKLGDLLNKTTIIFKPSSGGDPEITTHSFNCGG EFFYCNTSRLFNSTWNNTKWNSTGKITLPCRIKQIINMWQGVGKAMYAPPIEGLIKCSSNIT GLLLTRDGGANNSHNETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRAKRRWEREKRA IGLGAMFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIK QLQARILAVERYLKDQQLLGIWGCSGKHICTTTVPWNSSWSNKSLDEIWNNMTWMEWEREID NYTGLIYSLIEESQNQQEKNEQELLELDKWASLWNWFSITQWLWYIKIFIMIVGGLIGLRIV FAVLSLVNRVRQGYSPLSFQTLLPAPRGPDRPEGIEEEGGEQGRGRSIRLVNGFSALIWDDL RNLCLFSYHRLRDLILIAARIVELLGRRGWEALKYLWNLLQYWIQELKNSAI SLFDTTAIAV AEGTDRVIEIVQRACRAILNIPTRIRQGLERALL (SEQ ID NO: 27)

CONSENSUS. F MRVRGMQRNWQHLGKWGLLFLGILIICNAAENLWVTVYYGVPVWKEATTTLFCASDAKSYEK Fl A70C- EVHNVWCTHACVPTDPNPQEWLENVTENFDMWKNNMVEQMHTDII SCWDQSLKPCVKLTPL L111C CVTLNCTDVNATNNDTNDNKTGAIQNCSFNMTTEVRDKKLKVHALFYKLDIVPI SNNNSKYR

LINCNTSTITQACPKVSWDPIPIHYCAPAGYAILKCNDKRFNGTGPCKNVSTVQCTHGIKPV VSTQLLLNGSLAEEDI IIRSQNISDNAKTI IVHLNESVQINCTRPNNNTRKSIHLGPGQAFY ATGEIIGDIRKAHCNI SGTQWNKTLEQVKAKLKSHFPNKTIKFNSSSGGDLEITMHSFNCRG EFFYCNTSGLFNDTGSNGTITLPCRIKQIVNMWQEVGRAMYAAPIAGNITCNSNITGLLLTR DGGQNNTETFRPGGGNMKDNWRSELYKYKVVEIEPLGVAPTKAKRQWKRERRAVGIGAVFL GFLGAAGSTMGAASITLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLA VERYLKDQQLLGLWGCSGKLICTTNVPWNSSWSNKSQDEIWNNMTWMEWEKEISNYSNIIYR LIEESQNQQEKNEQELLALDKWASLWNWFDISNWLWYIKIFIMIVGGLIGLRIVFAVLSIVN RVRKGYSPLSLQTLIPSPREPDRPEGIEEGGGEQGKDRSVRLVNGFLALVWDDLRNLCLFSY RHLRDFILIAARIVDRGLRRGWEALKYLGNLTQYWSQELKNSAI SLLNTTAIWAEGTDRVI EALQRAGRAVLNIPRRIRQGLERALL (SEQ ID NO: 28)

CONSENSUS. F MRVREMQRNWQHLGKWGLLFLGILIICNAADNLWVTVYYGVPVWKEATTTLFCASDAKAYER F2 A70C- EVHNVWCTYACVPTDPSPQELVLGNVTENFNMWKNNMVDQMHEDII SCWDQSLKPCVKLTPL L111C CVTLNCTDVNVTINTTNVTLGEIKNCSFNITTEIKDKKKKEYALFYRLDVVPINNSIVYRLI

SCNTSTVTQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGLCRNVSTVQCTHGIRPVVS TQLLLNGSLAEEDI IIRSENISDNTKTI IVQFNRSVEINCTRPNNNTRKSIRIGPGRAFYAT GDIIGDIRKAYCNINRTLWNETLKKVAEEFKNHFNITVTFNPSSGGDLEITTHSFNCRGEFF YCNTSDLFNNTEVNNTKTITLPCRIRQFVNMWQRVGRAMYAPPIAGQIQCNSNITGLLLTRD GGKNGSETLRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRQVVQREKRAVGIGAVLLG FLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLKAIEAQQHLLQLTVWGIKQLQARILAV ERYLKDQQLLGIWGCSGKLICTTNVPWNSSWSNKSQDEIWDNMTWMQWEKEI SNYTDTIYRL IEDAQNQQEKNEQDLLALDKWDNLWSWFTITNWLWYIKIFIMIVGGLIGLRIVFAVLSWNR VRQGYSPLSLQTLIPNPRGPERPGGIEEEGGEQDRDRSIRLVSGFLALAWDDLRSLCLFSYR HLRDFILIAARTVDMGLKRGWEALKYLWNLPQYWGQELKNSAISLLDTTAIAVAEGTDRI IE VLQRAGRAVLHIPRRIRQGFERALL (SEQ ID NO: 29)

B. Isolated Peptides

Isolated peptides are disclosed herein comprising or consisting of amino acids of the CD4-BS2, such as an amino acid sequence including gpl20 positions 59-68 (HXB2 numbering), such as a peptide comprising or consisting of gpl20 positions 54-74. The disclosed HIV-1 neutralizing peptides are useful to inhibit or treat HIV-1 infection in vertebrate animals (such as mammals, for example, primates, such as humans). The disclosed HIV-1 neutralizing peptides are useful to induce immunogenic responses in vertebrate animals (such as mammals, for example, primates, such as humans) to HIV-1. Thus, In some embodiments, the disclosed HIV-1 neutralizing peptides are immunogens.

In some embodiments, the isolated peptide comprises or consists of gpl20 positions 59-68, wherein the HIV-1 neutralizing peptide includes the amino acid sequence set forth as KAYX₁X₂EVHNV (SEQ ID NO: 17), wherein Xi is aspartate or glutamate and X2 is threonine, lysine, or arginine; and wherein the peptide is no more than 50 amino acids in length. In some examples, the HIV-1 neutralizing peptide includes or consists of an amino acid sequence set forth as KAYDTEVHNV (SEQ ID NO: 18). and wherein the peptide is no more than 50 amino acids in length..

In additional embodiments, the isolated peptide comprises or consists of gpl20 positions 54-74, wherein the HIV-1 neutralizing peptide includes or consists of an amino acid sequence set forth as CASDAKAYX₁X₂EVHNVWATHAC, wherein Xi is aspartate or glutamate and X₂ is threonine, lysine, or arginine (SEQ ID NO: 19) wherein the peptide comprises a disulfide bond between the cysteine residues at positions 1 and 21; and wherein the peptide is no more than 50 amino acids in length. In some examples, the HIV-1 neutralizing peptide includes or consists of the amino acid sequence set forth as

CASDAKAYDTEVHNVWATHAC (SEQ ID NO: 20), wherein the peptide comprises a disulfide bond between the cysteine residues at positions 1 and 21; and wherein the peptide is no more than 50 amino acids in length.

In some embodiments, the isolated peptide can include the amino acid sequence of a gpl20 from an

HIV-1 strain, or an amino acid sequence at least 90% identical thereto. The isolated peptide can be derived from any subtype of HIV, such as groups M, N, O, or P or clade A, B, C, D, F, G, H, J or K and the like. For example, in some embodiments, the isolated peptide can comprise or consist of gpl20 residues 59-68 and comprise 10-50 consecutive amino acids (such as 10-15, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, or 20-50 consecutive amino acids) from a native HIV-1 gpl20 polypeptide sequence, such as a native HIV-1 gpl20 polypeptide sequence available in the HIV Sequence Database (hiv-web.lanl.gov/content hiv- db/mainpage.html), for example, a consensus HIV-1 gpl20 polypeptide sequence from genetic subtype A-F set forth in Table 1, or a polypeptide sequence at least 90% (for example, at least 95%, 96%, 97%, 98% or 99%) identical thereto. In some embodiments, the isolated peptide can comprise or consist of gpl20 residues 54-74 and comprise 21-50 consecutive amino acids (such as 21-30, or 21-40, or 20-50 consecutive amino acids) from a native HIV-1 gpl20 polypeptide sequence, such as a native HIV-1 gpl20 polypeptide sequence available in the HIV Sequence Database (hiv-web.lanl.gov/content hiv-db/mainpage.html), for example, a consensus HIV-1 gpl20 polypeptide sequence from genetic subtype A-F set forth in Table 1, or a polypeptide sequence at least 90% (for example, at least 95%, 96%, 97%, 98% or 99%) identical thereto.

In some embodiments, the HIV neutralizing peptide is also of a maximum length, for example, no more than 10, 11, 12, 13, 14, 15, 20, 30, 40, or 50 amino acids amino acids in length. The HIV neutralizing peptide may include, consist or consist essentially of the disclosed sequences. The disclosed contiguous sequences may also be joined at either end to other unrelated sequences (for example, non-gpl20, non-HIV- 1, non-viral envelope, or non-viral protein sequences).

Several embodiments include a multimer of any of the disclosed peptides, for example, a multimer including 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of the disclosed HIV-1 neutralizing peptides. In some examples, any of the disclosed HIV-1 neutralizing peptides can be linked to another of the disclosed HIV-1 neutralizing peptides to form the multimer.

In some embodiments, the HIV-neutralizing peptide is linked to a heterologous scaffold derived from another protein of human, animal, vegetal or synthetic origin, which serves to either stabilize its structure, increase its potency, or improve its pharmacological properties such as plasma half-life or resistance to protease digestion. In other examples a single scaffold can bind multiple copies of the neutralizing peptide. Examples of scaffold proteins include (but are not limited to) tetanus toxoid, cholera toxin beta-subunit, albumin, or the Fc portion of human immunoglobulin (Ig)G or IgM.

In some embodiments, the isolated peptide can bind to CD4. In some examples, the dissociation constant for CD4 binding to the HIV-1 neutralizing peptide, is less than about 10^"4 Molar, such as less than about 10^"5 Molar, 10^"6 Molar, 10^"7 Molar, or less than 10^"8 Molar. Binding to CD4 can be determined by methods known in the art. The determination that a particular agent binds substantially only to a specific polypeptide may readily be made by using or adapting routine procedures.

In some embodiments, the isolated peptide can include an alpha helical structure. For example, gpl20 positions 59-68 of the isolated peptide can include an alpha helical structure. Conventional methods (such as circular dichroism measurements) can be used to determine if the peptide includes the alpha helical structure.

In some embodiments, any of the isolated peptides can be used to elicit an immune response to HIV- 1 in a subject. In some such embodiments, induction of the immune response includes production of broadly neutralizing antibodies to HIV-1. Methods to assay for neutralization activity are known to the person of ordinary skill in the art and are further described herein. Standard methods in the art can be used to make the disclosed peptides. For example, recombinant DNA technology can be used to generate a nucleic acid encoding the disclosed peptides, and from which the peptide can be expressed and purified. Such methods are known to the skilled artisan and further described herein. In addition to recombinant methods, the peptides that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the peptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc.

85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Π1., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicylohexylcarbodimide) are well known in the art. Methods of generating a peptide with one or more sulfated tyrosine residues are known to the person of ordinary skill in the art, and described herein (see, e.g., U.S. Pub. No. 5,541,095, 2009/0042738, 2006/0009631,

2003/0170849, 2006/0115859, and Liu et al, Mol. Biosyst., 7:38-47, 2011, each of which is incorporated by reference herein).

C. Polynucleotides and Expression

Polynucleotides encoding a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4- BS2) are also provided. These polynucleotides include DNA, cDNA and RNA sequences which encode the antigen. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the nucleic acid sequence.

In some embodiments, the nucleic acid molecule encodes a precursor of a disclosed recombinant HIV-1 Env ectodomain or immunogenic fragment thereof, that, when expressed in an appropriate cell, is processed into a disclosed recombinant HIV-1 Env ectodomain or immunogenic fragment thereof. For example, the nucleic acid molecule can encode a recombinant HIV-1 Env ectodomain including a N- terminal signal sequence for entry into the cellular secretory system that is proteolytically cleaved in the during processing of the HIV-1 Env protein in the cell. In some embodiments, the signal peptide includes the amino acid sequence set forth as residues 1-30 of SEQ ID NO: 2.

Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^th ed, Cold

Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

The polynucleotides encoding a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4- BS2) can include a recombinant DNA which is incorporated into a vector into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.

Polynucleotide sequences encoding a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gp l20, or an isolated peptide including residues of the CD4-BS2) can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

DNA sequences encoding the disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4- BS2) can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, e.g., Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4^th Ed., Humana Press). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some embodiments, the host cells include HEK293 cells or derivatives thereof, such as GnTI^_/" cells (ATCC® No. CRL-3022), or HEK-293F cells.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC method using procedures well known in the art. Alternatively, MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

In one non-limiting example, a disclosed immunogen is expressed using the pVRC8400 vector (described in Barouch et al., J. Virol, 79 ,8828-8834, 2005, which is incorporated by reference herein).

Modifications can be made to a nucleic acid encoding a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4-BS2) without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.

In addition to recombinant methods, the recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4-BS2 can also be constructed in whole or in part using protein synthesis methods known in the art. D. Viral Vectors

A nucleic acid molecule encoding a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4- BS2) can be included in a viral vector, for example, for expression of the immunogen in a host cell, or for immunization of a subject as disclosed herein. In some embodiments, the viral vectors are administered to a subject as part of a prime-boost vaccination. In some embodiments, the viral vectors are included in a vaccine, such as a primer vaccine or a booster vaccine for use in a prime-boost vaccination.

In some examples, the viral vector can be replication-competent. For example, the viral vector can have a mutation in the viral genome that does not inhibit viral replication in host cells. The viral vector also can be conditionally replication-competent. In other examples, the viral vector is replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used to express the disclosed antigens, including polyoma, i.e., SV40 (Madzak et al., 1992, /. Gen. Virol., 73: 15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al, 1992, /. Virol., 66:4407-4412; Quantin et al, 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584;

Rosenfeld et al, 1992, Cell, 68: 143-155; Wilkinson et al, 1992, Nucl. Acids Res., 20:2233-2239; Stratford- Perricaudet et al, 1990, Hum. Gene Ther, 1 :241-256), vaccinia virus (Mackett et al, 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol, 158:91-123; On et al, 1990, Gene, 89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.

Microbiol. Immunol, 158:67-90; Johnson et al, 1992, /. Virol, 66:29522965; Fink et al, 1992, Hum. Gene Ther. 3: 11-19; Breakfield et al, 1987, Mol. NeurobioL, 1:337-371; Fresse et al, 1990, Biochem.

Pharmacol, 40:2189-2199), Sindbis viruses (H. Herweijer et al, 1995, Human Gene Therapy 6: 1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11: 18-22; I. Frolov et al, 1996, Proc. Natl. Acad. Sci. USA 93: 11371-11377) and retroviruses of avian

(Brandyopadhyay et al, 1984, Mol. Cell Biol, 4:749-754; Petropouplos et al, 1992, /. Virol, 66:3391-

3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol, 158: 1-24; Miller et al. , 1985, Mol. Cell Biol, 5:431-437; Sorge et al, 1984, Mol. Cell Biol, 4:1730-1737; Mann et al, 1985, /. Virol, 54:401-407), and human origin (Page et al, 1990, /. Virol, 64:5370-5276; Buchschalcher et al, 1992, /. Virol, 66:2731- 2739). Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

In some embodiments, the viral vector can include an adenoviral vector that expresses a disclosed recombinant HIV-1 Env ectodomain or immunogenic fragment thereof. Adenovirus from various origins, subtypes, or mixture of subtypes can be used as the source of the viral genome for the adenoviral vector. Non-human adenovirus (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can be used to generate the adenoviral vector. For example, a simian adenovirus can be used as the source of the viral genome of the adenoviral vector. A simian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. A simian adenovirus can be referred to by using any suitable abbreviation known in the art, such as, for example, SV, SAdV, SAV or sAV. In some examples, a simian adenoviral vector is a simian adenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27: 1293- 1300, 2009). Human adenovirus can be used as the source of the viral genome for the adenoviral vector. Human adenovirus can be of various subgroups or serotypes. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype. The person of ordinary skill in the art is familiar with replication competent and deficient adenoviral vectors (including singly and multiply replication deficient adenoviral vectors). Examples of replication-deficient adenoviral vectors, including multiply replication-deficient adenoviral vectors, are disclosed in U.S. Patent Nos. 5,837,51 1 ; 5,851 ,806; 5,994, 106; 6, 127, 175; 6,482,616; and 7, 195,896, and International Patent Application Nos. WO 94/28152, WO 95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO

97/12986, WO 97/21826, and WO 03/02231 1.

E. Pharmaceutical Compositions

Immunogenic compositions comprising a disclosed immunogen (e.g., a recombinant gpl20, a HIV-1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4-BS2) and a pharmaceutically acceptable carrier are also provided. Such pharmaceutical compositions can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In some embodiments, pharmaceutical compositions including one or more of the disclosed immunogens are immunogenic compositions. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

Thus, a immunogen described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing. Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually≤1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The pharmaceutical compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

The pharmaceutical composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, A1P04, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxy ethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12

(Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89- 142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

In some instances it may be desirable to combine a disclosed immunogen, with other pharmaceutical products (e.g., vaccines) which induce protective responses to other agents. For example, a composition including a recombinant paramyxovirus as described herein can be can be administered simultaneously (typically separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html) for the targeted age group (e.g., infants from approximately one to six months of age). As such, a dicalosed immunogen including a recombinant HIV-1 gpl20 described herein may be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio, influenza and rotavirus.

In some embodiments, the composition can be provided as a sterile composition. The

pharmaceutical composition typically contains an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the

immunogenic composition is selected as an amount which induces an immune response without significant, adverse side effects. In some embodiments, the composition can be provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent HIV-1 infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof. In other embodiments, the composition further includes an adjuvant.

III. Therapeutic Methods

The disclosed immunogens (e.g., a recombinant gpl20, a HIV- 1 Env ectodomain trimer comprising the recombinant gpl20, or an isolated peptide including residues of the CD4-BS2), polynucleotides and vectors encoding the disclosed immunogens, and compositions including same, can be used in methods of preventing, inhibiting and treating an HIV- 1 infection, as well as methods of eliciting an immune response to HIV-1. In some embodiments, a therapeutically effective amount of an immunogenic composition including one or more of the disclosed immunogens, can be administered to a subject in order to generate an immune response to HIV-1.

When inhibiting, treating, or preventing HIV- 1 infection, the methods can be used either to avoid infection in an HIV- 1 seronegative subject (e.g., by eliciting an immune response that protects against HIV- 1 infection), or to treat existing infection in an HIV-1 seropositive subject. The HIV-1 seropositive subject may or may not carry a diagnosis of AIDS. Hence in some embodiments the methods involves selecting a subject at risk for contracting HIV-1 infection, or a subject at risk of developing AIDS (such as a subject with HIV-1 infection), and administering a disclosed immunogen to the subject to elicit an immune response to HIV-1 in the subject.

Treatment of HIV-1 by inhibiting HIV-1 replication or infection can include delaying the development of AIDS in a subject. Treatment of HIV- 1 can also include reducing signs or symptoms associated with the presence of HIV-1 (for example, by reducing or inhibiting HIV-1 replication). In some examples, treatment using the methods disclosed herein prolongs the time of survival of the subject.

Typical subjects intended for treatment with the therapeutics and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional workups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods, which are available and well known in the art to detect and/or characterize HIV-1 infection. These and other routine methods allow the clinician to select patients in need of therapy using the methods and pharmaceutical compositions of the disclosure. In accordance with these methods and principles, a composition can be administered according to the teachings herein, or other conventional methods known to the person of ordinary skill in the art, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

The administration of a disclosed immunogen can be for prophylactic or therapeutic purpose. When provided prophylactically, the disclosed therapeutic agents are provided in advance of any symptom, for example, in advance of infection. The prophylactic administration of the disclosed therapeutic agents serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the disclosed therapeutic agents are provided at or after the onset of a symptom of disease or infection, for example, after development of a symptom of HIV- 1 infection, or after diagnosis of HIV- 1 infection. The therapeutic agents can thus be provided prior to the anticipated exposure to HIV-1 virus so as to attenuate the anticipated severity, duration or extent of an infection and/or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection.

The immunogenic composition including one or more of the disclosed immunogens, can be used in coordinate (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-HIV-1 immune response, such as an immune response to HIV-1 Env protein. Separate immunogenic compositions that elicit the anti-HIV-1 immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic

compositions) in a coordinate immunization protocol.

In one embodiment, a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions including a disclosed immunogen, with a second inoculation being administered more than about two, about three to eight, or about four, weeks following the first inoculation. A third inoculation can be administered several months after the second inoculation, and in specific embodiments, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's "immune memory." The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of HIV-1 infection or progression to AIDS, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency to an uninfected partner. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the disclosed recombinant HIV-1 Env protein, immunogenic fragment thereof, protein nanoparticle, polynucleotide encoding a recombinant HIV- 1 Env ectodomain or immunogenic fragment, vector or composition and/or adjuvant can be increased or the route of administration can be changed.

It is contemplated that there can be several boosts, and that each boost can be a different disclosed immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime. The prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. The immune response against the selected antigenic surface can be generated by one or more inoculations of a subject.

Upon administration of a disclosed immunogen of this disclosure, the immune system of the subject typically responds to the immunogenic composition by producing antibodies specific for HIV-1 Env protein. Such a response signifies that an immunologically effective dose was delivered to the subject.

An immunologically effective dosage can be achieved by single or multiple administrations

(including, for example, multiple administrations per day), daily, or weekly administrations. For each particular subject, specific dosage regimens can be evaluated and adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the immunogenic composition. In some embodiments, the antibody response of a subject will be determined in the context of evaluating effective dosages/immunization protocols. In most instances it will be sufficient to assess the antibody titer in serum or plasma obtained from the subject. Decisions as to whether to administer booster inoculations and/or to change the amount of the therapeutic agent administered to the individual can be at least partially based on the antibody titer level. The antibody titer level can be based on, for example, an immunobinding assay which measures the concentration of antibodies in the serum which bind to an antigen including, for example, a disclosed recombinant HIV-1 Env protein. The methods of using immunogenic composition, and the related compositions and methods of the disclosure are useful in increasing resistance to, inhibiting preventing, ameliorating, and/or treating infection and disease caused by HIV-1 in animal hosts, and other, in vitro applications.

In some embodiments, a disclosed immunogen can be administered to the subject simultaneously with the administration of an adjuvant. In other embodiments, the immunogen can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to induce the immune response.

Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject, or that induce a desired response in the subject (such as a neutralizing immune response). Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the composition (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. The actual dosage of disclosed immunogen will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. As described above in the forgoing listing of terms, a therapeutically effective amount is also one in which any toxic or detrimental side effects of the disclosed immunogen and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects.

A non-limiting range for a therapeutically effective amount of the disclosed immunogen within the methods and immunogenic compositions of the disclosure is about 0.0001 mg/kg body weight to about 10 mg/kg body weight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, or about 10 mg/kg, for example, 0.01 mg/kg to about 1 mg/kg body weight, about 0.05 mg/kg to about 5 mg/kg body weight, about 0.2 mg/kg to about 2 mg/kg body weight, or about 1.0 mg/kg to about 10 mg/kg body weight. In some embodiments, the dosage includes a set amount of a disclosed immunogen such as from about 1-300 μg, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 μg.

The dosage and number of doses will depend on the setting, for example, in an adult or anyone primed by prior HIV-1 infection or immunization, a single dose may be a sufficient booster. In naive subjects, in some examples, at least two doses would be given, for example, at least three doses. In some embodiments, an annual boost is given, for example, along with an annual influenza vaccination.

Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

In some embodiments, it may be advantageous to administer the therapeutic agents disclosed herein with other agents such as proteins, peptides, antibodies, and other antiviral agents, such as anti-HIV-1 agents. Examples of such anti-HIV-1 therapeutic agents include nucleoside reverse transcriptase inhibitors, such as abacavir, AZT, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, and the like, non-nucleoside reverse transcriptase inhibitors, such as delavirdine, efavirenz, nevirapine, protease inhibitors such as amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, osamprenavir, ritonavir, saquinavir, tipranavir, and the like, and fusion protein inhibitors such as enfuvirtide and the like. In some examples, the disclosed therapeutic agents are administered with T-helper cells, such as exogenous T-helper cells. Exemplary methods for producing and administering T-helper cells can be found in International Patent Publication WO 03/020904, which is incorporated herein by reference.

For any application, treatment with a disclosed immunogen can be combined with anti-retroviral therapy, such as HAART. Antiretroviral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The therapeutic agents can be administered before, during, concurrent to and/or after retroviral therapy. In some embodiments, the therapeutic agents are administered following a course of retroviral therapy. The disclosed therapeutic agents can be administered in conjunction with nucleoside and nucleotide reverse transcriptase inhibitors (nRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors, Entry inhibitors (or fusion inhibitors), Maturation inhibitors, or a broad spectrum inhibitors, such as natural antivirals. Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine and efavirenz.

In some embodiments, the disclosed immunogen is administered to the subject simultaneously with the administration of the adjuvant. In other embodiments, the disclosed immunogen is administered to the subject after the administration of the adjuvant and within a sufficient amount of time to induce the immune response.

HIV-1 infection does not need to be completely eliminated or reduced or prevented for the methods to be effective. For example, elicitation of an immune response to HIV-1 with one or more of the disclosed immunogens can reduce or inhibit HIV-1 infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HIV-1 infected cells), as compared to HIV-1 infection in the absence of the therapeutic agent. In additional examples, HIV-1 replication can be reduced or inhibited by the disclosed methods. HIV-1 replication does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed immunogens can reduce HIV-1 replication by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HIV-1 replication), as compared to HIV-1 replication in the absence of the immune response.

To successfully reproduce itself, HIV-1 must convert its RNA genome to DNA, which is then imported into the host cell's nucleus and inserted into the host genome through the action of HIV-1 integrase. Because HIV-l's primary cellular target, CD4+ T-Cells, can function as the memory cells of the immune system, integrated HIV-1 can remain dormant for the duration of these cells' lifetime. Memory T- Cells may survive for many years and possibly for decades. This latent HIV-1 reservoir can be measured by co-culturing CD4+ T-Cells from infected patients with CD4+ T-Cells from uninfected donors and measuring HIV-1 protein or RNA (See, e.g., Archin et al, AIDS, 22: 1131-1135, 2008). In some embodiments, the provided methods of treating or inhibiting HIV-1 infection include reduction or elimination of the latent reservoir of HIV-1 infected cells in a subject. For example, a reduction of at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV-1) of the latent reservoir of HIV-1 infected cells in a subject, as compared to the latent reservoir of HIV- 1 infected cells in a subject in the absence of the treatment with one or more of the provided immunogens.

Studies have shown that the rate of HIV-1 transmission from mother to infant is reduced significantly when zidovudine is administered to HIV-1 -infected women during pregnancy and delivery and to the offspring after birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541). Several studies of mother - to-infant transmission of HIV- 1 have demonstrated a correlation between the maternal virus load at delivery and risk of HIV-1 transmission to the child. The disclosed immunogens are of use in decreasing HIV- 1- transmission from mother to infant. Thus, in some embodiments a therapeutically effective amount of one or more of the provided therapeutic agents is administered in order to prevent transmission of HIV- 1, or decrease the risk of transmission of HIV- 1, from a mother to an infant. In some embodiments, a therapeutically effective amount of the agent can be administered to a pregnant subject to elicit an immune response that generates neutralizing antibodies that are passes to the fetus via the umbilical cord to protect the fetus from infection during birth. In some embodiments, both a therapeutically effective amount of a disclosed immunogen and a therapeutically effective amount of another anti-HIV- 1 agent, such as zidovudine, is administered to the mother and/or infant.

Following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity are known to the person of ordinary skill in the art and are further described herein, and include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays (e.g., as described in Martin et al. (2003) Nature Biotechnology 21 :71-76), and pseudo virus neutralization assays (e.g., as described in Georgiev et al. (Science, 340, 751-756, 2013),

Seaman et al. (J. Virol., 84, 1439- 1452, 2005), and Mascola et al. (J. Virol., 79, 10103-10107, 2005), each of which is incorporated by reference herein in its entirety. In some embodiments, the serum neutralization activity can be assayed using a panel of HIV-1 pseudoviruses as described in Georgiev et al., Science, 340, 751-756, 2013 or Seaman et al. J. Virol., 84, 1439-1452, 2005. Briefly, pseudovirus stocks are prepared by co-transfection of 293T cells with an HIV-1 Env-deficient backbone and an expression plasmid encoding the Env gene of interest. The serum to be assayed is diluted in Dulbecco's modified Eagle medium- 10% FCS (Gibco) and mixed with pseudovirus. After 30 min, 10,000 TZM-bl cells are added, and the plates are incubated for 48 hours. Assays are developed with a luciferase assay system (Promega, Madison, WI), and the relative light units (RLU) are read on a lumino meter (Perkin-Elmer, Waltham, MA). To account for background, a cutoff of ID₅₀ > 40 can be used as a criterion for the presence of serum neutralization activity against a given pseudovirus. In some embodiments, administration of a therapeutically effective amount of one or more of the disclosed immunogen to a subject (e.g., by a prime-boost administration of a DNA vector encoding a disclosed immunogen (prime) followed by a protein nanoparticle including a disclosed immunogen (boost)) induces a neutralizing immune response in the subject, wherein serum from the subject neutralizes, with an ID50 > 40, at least 30% (such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of pseudoviruses is a panel of pseudo viruses including the HIV-1 Env proteins listed in Table S5 or Table S6 of Georgiev et al. (Science, 340, 751-756, 2013), or Table 1 of Seaman et al. (J. Virol., 84, 1439-1452, 2005).

One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No. 5,880, 103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune-stimulating constructs, or ISCOMS™, negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and Quil A™ (saponin). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein- Barr virus-induced tumors, using ISCOMS™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™ have been found to produce Class I mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

In some embodiments, a plasmid DNA vaccine is used to express a disclosed immunogen in a subject. For example, a nucleic acid molecule encoding a disclosed immunogen can be administered to a subject to elicit an immune response to HIV-1 gpl20. In some embodiments, the nucleic acid molecule can be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated by reference herein).

In another approach to using nucleic acids for immunization, a disclosed recombinant gpl20 or HIV-1 Env ectodomain trimer can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytogmeglo virus or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351 :456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed recombinant gpl20 or HIV-1 Env ectodomain trimer is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad' s HELIOS™ Gene Gun. The nucleic acids can be "naked," consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites.

Dosages for injection are usually around 0.5 μg/kg to about 50 mg kg, and typically are about 0.005 mg kg to about 5 mg kg (see, e.g., U.S. Patent No. 5,589,466).

EXAMPLES

The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified. Example 1

Quaternary Configuration of the HIV-1 Receptor-Binding Site

This example illustrates that the HIV-1 Env ectodomain timer interacts with CD4 through a quaternary surface formed by coalescence of the previously defined CD4-contact region in the gpl20 outer domain (CD4-BS1) with a second binding site (CD4-BS2) in a neighboring gpl20 protomer, which encompasses CI and C2 elements from the inner domain. Disruption of CD4-BS2 reduces the stability of CD4-trimer interaction and abrogates HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurs upon mutation of CD4 residues that interact with CD4-BS2. Selected neutralizing human antibodies also recognize a quaternary surface that spans CD4-BS2, providing evidence that this region is immunogenic in vivo. These results illustrate the quaternary configuration of a retroviral receptor-binding site, and allow design of novel HIV- 1 Env based therapeutic agents and immunogens.

The native trimeric HIV-1 Env spike displayed on the surface of mature virions is the sole functional form that mediates viral attachment and entry. Interaction of conserved elements in the external envelope glycoprotein, gpl20, with the CD4 receptor is the first critical step in the HIV-1 infectious cycle (Dalgleish et al, Nature, 312, 763-767, 1984; Klatzmann et al, Nature, 312, 767-768, 1984). Although the CD4- binding site in gpl20 has been extensively characterized by both mutagenesis (Lasky et al, Cell, 50, 975- 985, 1987; Olshevsky et al, J. Virol, 64, 5701-5707, 1990; Pantophlet et al, Virol, 77, 642-658, 2003; Finzi et al, Mol. Cell, 37, 656-667, 2010 and co-crystallization with sCD4 (Kwong et al, Nature, 393, 648- 659, 1998; Kwong et al, Structure, 8, 1329-1339, 2000; Huang et al, Science, 310, 1025-1028, 2005; Pancera et al, PNAS, 107, 1166-1171, 2010), most of these studies were performed on monomeric gpl20 subunits, thereby precluding the recognition of quaternary structure-specific components.

The availability of high-resolution structures from a soluble cleaved subtype- A gpl40 trimer, BG505-SOSIP.664 (Julien et al, Science, 342, 1477-1483, 2013; Pancera et al, Nature, 514, 455-461, 2014; Kwon et al, Nat. Struct. Mol. Biol, 22, 522-531, 2015), has offered the opportunity to evaluate the role of quaternary interactions of the HIV-1 envelope with the CD4 receptor. A 2-domain sCD4 was docked to a trimer structure (PDB ID: 4TVP) (Pancera et al, Nature, 514, 455-461, 2014) by aligning a sCD4- bound monomeric gpl20 structure, inclusive of the N-terminal region (PDB ID: 3JWD) (Pancera et al, PNAS, 107, 1166-1171, 2010), to one gpl20 protomer of the trimer followed by energy minimization (FIGs. 1A and IB). The alignment shows domain 1 of CD4 reaching deep into the interprotomer groove and establishing contacts not only with the previously defined CD4-binding site (CD4-BS) in the gpl20 outer domain (Lasky et al, Cell, 50, 975-985, 1987; Olshevsky et al. J. Virol, 64, 5701-5707, 1990; Pantophlet et al, J. Virol, 77, 642-658, 2003; Finzi et al, Mol. Cell, 37, 656-667, 2010; Kwong et al, Nature, 393, 648- 659, 1998; Kwong et al, Structure, 8, 1329-1339, 2000; Huang et al, Science, 310, 1025-1028, 2005; Pancera et al, PNAS, 107, 1166-1171, 2010), but also with a second binding site (CD4-BS2) located in the inner domain of a neighboring gpl20 protomer, which encompasses amino acid residues in the CI region (E62, T63, E64 and H66 in the -1 helix) and the C2 region (K207 at the base of the β3-β4 loop). Thus, the extended CD4-contact surface appears like an oblong cavity formed by coalescence of discontinuous domains on two adjacent gpl20 protomers (FIG. 5A). Residues E64, H66 and K207 are nearly universally conserved (>99.7%) across all group-M HIV-1 isolates, while position 62 is occupied by an acidic residue (E or D) in more than 90% of group-M isolates (FIG. 5B).

To assess the plausibility and stability of the interaction of CD4 with CD4-BS2, extensive molecular dynamics (MD) simulations of the soluble trimer in complex with sCD4 were conducted, which showed the interaction to be stable over time (Table 2). Two-domain sCD4 was docked to the structure of a soluble cleaved HIV-1 gpl40 trimer (PDB ID: 4TVP) by aligning a sCD4-bound monomeric gpl20 structure, inclusive of the N- terminal region (PDB ID: 3JWD), to one gpl20 protomer of the trimer followed by energy minimization. MD analysis was performed on a multimolecular complex formed by three sCD4 molecules bound to a soluble cleaved HIV-1 Env trimer (BG505-SOSIP.664). All three CD4 proteins bound to the trimer were examined and their interaction with CD4-BS2 was stable over a prolonged (172 nsec) MD simulation. Area values for each sCD4 molecule contact with the CD4-BS 1 and CD4-BS2 regions were calculated in the three lowest-energy frames in the MD simulation. The values denote the contact area within 3 A of sCD4.

Table 2. Contact surface between CD4 and two CD4-binding sites located in two distinct domains of HIV-1 gpl20

CD4-BS1 (A²) CD4-BS2 (A²) Total (A²)

Frame 65.2

CD4_a 1134.46 288.23 1422.69

CD4_b 1114.09 118.86 1232.95

CD4_c 837.53 371.05 1208.58

Average 1028.69 259.38 1288.07

Frame 146.4

CD4_a 1077.83 203.08 1280.91

CD4_b 940.57 115.83 1056.40

CD4_c 785.12 245.14 1030.26

Average 934.51 188.02 1122.52

Frame 1192.5

CD4_a 1250.60 110.04 1360.64 CD4_b 897.01 113.79 1010.80

CD4_c 739.06 124.02 863.08

Average 962.22 115.95 1078.17

To investigate the functional relevance of CD4-BS2 to HIV-1 infection, the residues E62, E64, H66, and K207 were mutagenized in full-length gpl60 from two different isolates, BG505 (clade A) and BaL (clade B), to generate infectious viral pseudoparticles. Both individual and combined charge inversions were introduced to create a reversely-charged surface; as a control, a key residue (D368) of the classic CD4- BS was mutated (Olshevsky et al, J. Virol, 64, 5701-5707, 1990). Both WT and mutated gpl60 were efficiently expressed on the surface membrane of transfected human embryonic kidney (HEK) 293T cells and displayed correct folding and trimeric structure, as determined by reactivity with a panel of anti- envelope monoclonal antibodies (mAbs) including the trimer-preferring PG9, 35022 and PGT151 (FIGs. 6A and 6B). Charge inversions of E62/E64, H66 and K207 completely abrogated the infectivity of both

HIV-1 envelopes, as did the control D368R mutant, whereas charge inversion of neighboring basic residues (K65 in BG505; K59 in BaL) had limited or no effects (FIG. 1C). The loss of infectivity was not due to reduced gpl20-gp41 association, as shown by unaltered levels of gpl20 shedding (FIGs. 7A and 7B). A set of additional mutations was introduced into the BaL envelope in order to get a more complete topological and functional mapping of the CD4-BS2 region (FIG. 8). Alanine substitutions of E62, E64, H66, and K207, either individually or in combination, consistently reduced HIV-1 infectivity, albeit less markedly than charge inversions; however, a triple alanine mutant (E62A/E64A/K207A) showed a complete loss of infectious capacity. Charge inversion or alanine substitution of other solvent-exposed residues in the inner domain had no effects (K59E, E102A/K) or even enhanced infectivity (Y61A), corroborating the specificity of the effects observed upon disruption of CD4-BS2. These results demonstrate that interaction of CD4 with the complete quaternary contact surface on the HIV- 1 Env trimer is critical for the progression of the fusogenic process.

To elucidate the mechanism underlying the complete loss of infectivity induced by disruption of the CD4-BS2 region, the same residues (E62, E64, H66 and K207) were mutagenized in the BG505-SOSIP.664 soluble trimer, which displays an antigenic profile similar to that of the pre-fusion HIV-1 Env spike (Sanders et al, PLoS Pathog., 9, el003618, 2013). The WT and mutated trimers were expressed in HEK 293FS cells and purified to homogeneity. All the trimers displayed correct folding and trimeric configuration, as shown by reactivity with a wide panel of mAbs (FIGs. 9A and 9B). Mutation of key residues in CD4-BS2 markedly reduced the ability of the soluble trimer to bind sCD4 in ELISA (FIG. 2A), as well as native CD4 expressed on the surface of human T cells (FIG. 2B). By SPR analysis, all the CD4-BS2 mutants showed a reduced sCD4-binding affinity (KD), with K207E exhibiting the most pronounced decrease (KD= 93.7 nM compared to 4.6 nM for the WT), followed by H66E (K_D=66.5 nM) and E62K/E64K (K_D=49.3 nM); this effect was essentially due to a faster off -rate (kd), while the on-rate (k_a) was only marginally affected (FIG. 2C). Altogether, these results demonstrate that CD4-BS2 plays an important role in stabilizing the interaction of the HIV-1 Env trimer with CD4. The same mutations were introduced into monomeric gpl20 derived from both BG505 and BaL, and the proteins were expressed in HEK 293FS cells. Reactivity with reference anti-gpl20 mAbs confirmed the correct folding of the mutants (FIGs. 10B and IOC). None of the mutations had significant effects on sCD4 binding to monomeric gpl20 in ELISA (FIG. 10A). Of note, the CD4-BS2 region is completely disassembled in the monomeric structure due to the opposite orientation of the E62, E64, H66 and K207 side-chains, which point outwards and are fully solvated in the trimer (Kwon et al, Nat. Struct. Mol. Biol., 22, 522-531, 2015), while they point inwards and are buried within the core of the protein in the monomer (Kwong et al., Nature, 393, 648-659, 1998; Kwong et al., Structure, 8, 1329- 1339, 2000; Huang et al, Science, 310, 1025-1028, 2005; Pancera et al, PNAS, 107, 1166-1171, 2010) (FIG. 11).

Next, the events downstream of CD4 binding, which induce gpl20 to acquire corecep tor-binding competence and eventually dissociate from gp41, leading to activation of the fusogenic process were assayed. While in the absence of sCD4 the BG505-SOSIP.664 trimer was unable to bind to cell surface- expressed CCR5 by flow cytometry, efficient binding was detected after pre-activation with sCD4; in contrast, none of the CD4-BS2 mutants were able to bind to CCR5 regardless of sCD4 treatment (FIG. 2D). Likewise, no induction of the 48d epitope, which extensively overlaps the CCR5-binding surface (Xiang et al, AIDS Res. Hum. Retroviruses, 18, 1207-1217, 2002), was detected in any of the CD4-BS2 mutants upon treatment with sCD4 (FIG. 2E). These results indicate that interaction of CD4 with the inner domain is an essential step for triggering gpl20 conformational transitions that lead to coreceptor binding and progression of the envelope-mediated fusogenic process.

To further validate the role of CD4-BS2 in the envelope-receptor interaction, the residues in CD4 that make contact with CD4-BS2 were identified. Four residues in CD4 domain 1, never previously reported to contact gpl20 (Kwong et al, Nature, 393, 648-659, 1998; Kwong et al, Structure, 8, 1329- 1339, 2000; Huang et al, Science, 310, 1025-1028, 2005; Pancera et al. , PNAS, 107, 1166-1171, 2010), were identified as potentially interactive with CD4-BS2 by docking and MD simulations (E13 in strand B, K21 and K22 in the CDRl-like loop, and D63 in the DE loop) (FIG. 3A); each of these residues was mutagenized by charge inversion, and full-length CD4 mutants were expressed on the surface of HEK 293T cells and tested for HIV-1 receptor function. The correct expression and folding of the mutants was verified using a panel of anti-CD4 mAbs (FIG. 12B). Mutation of three residues, E13, K22 and D63, caused a dramatic loss of HIV-1 receptor function, as seen with both the BG505 and BaL envelopes, while charge inversion of K21 had more limited effects and alanine substitution of Q64 had no effects; as expected, the control F43A substitution caused a complete loss of receptor function (FIG. 3B). Analysis of a more extended panel of CD4 mutants showed that alanine substitutions of E13, K22 and D63 also reduced HIV-1 infectivity, albeit less markedly than charge inversions (FIG. 12A). These results demonstrate that alterations on both sides of the CD4/CD4-BS2 interface can disrupt the functional interaction between the HIV-1 Env and the CD4 receptor.

To assess the immunogenicity of CD4-BS2 and its relevance to antibody-mediated neutralization and immune evasion, whether disruption of CD4-BS2 could affect binding of several anti-CD4-BS human mAbs isolated from HIV- 1 -infected individuals was examined. Docking onto the trimer structure (PDB ID: 4TVP)(Pancera et al, Nature, 514, 455-461, 2014) showed that all these mAbs reach deep into the inter- protomer groove, but only some of them (most notably, VRC03, VRC06, VRC-CH31) appear to make direct contact with CD4-BS2 (FIG. 4A). Mutation of key residues in CD4-BS2 had diverse effects on binding of these mAbs to the BG505-SOSIP.664 trimer (FIG. 4B): charge inversion of K207 significantly decreased recognition by VRC03 and, to a lesser extent, by VRC-CH31, while mutation of E62/E64 selectively affected VRC06; none of these mutations significantly reduced binding of VRCOl, which does not appear to directly contact CD4-BS2. The diverse effects of CD4-BS2 mutations on VRC03 and VRC06 binding were confirmed by SPR analysis (FIG. 13A). These discrepant results suggest that none of the anti-CD4-BS mAbs precisely mimics the binding mode of CD4. A more extended panel of mAbs and envelope mutants was tested on cell-surface expressed gpl60 from both BG505 and BaL. As seen with the soluble trimer, the most pronounced effects were induced by the K207E mutation on VRC03, and by the E62K/E64K mutation on VRC06; recognition of cell-surface gpl60 by VRC06 and PG04 was also affected by mutations of K207 (FIGs. 14A-14D). In accordance with their dependence of CD4-BS2, both VRC03 and VRC06 exhibited a trimer-specific reactivity in ELISA (FIG. 13B). These results are consistent with the concealment of the key CD4-BS2 residues in monomeric gpl20 (FIG. 11), a strategy that may facilitate immune evasion by HIV-1. Altogether, these data show that certain neutralizing human mAbs establish quaternary contacts with the functional HIV-1 Env spike, demonstrating that the CD4-BS2 region is immunogenic in vivo.

This work provides evidence that the HIV-1 receptor-binding site has a quaternary configuration, a feature previously recognized for Picornaviruses (Xing et al, EMBO J., 19, 1207-1216, 2000; Neumann et al, J. Virol, 77, 8504-8511, 2003; Xing et al, J. Biol. Chem., 279, 11632-81163, 2004) but never described for enveloped viruses. The complete CD4-binding site of HIV-1 spans conserved regions from two neighboring gpl20 protomers, which converge to form an extended surface complementary to discontinuous elements in domain 1 of CD4. Putative quaternary interactions with CD4 or selected mAbs had previously been suggested by in silico alignment with a cryoEM trimer reconstruction, but the interactive surface was not defined, nor was its functional significance (Lyumkis et al, Science, 342, 1484-1490, 2013). The complete loss of infectivity induced by mutation of key residues on both sides of the interface indicates that contact of CD4 with CD4-BS2 is critical for the functional interaction between HIV- 1 and the CD4 receptor. While the classic CD4-BS in the outer domain of gpl20 appears to be responsible for initial receptor docking, as suggested by the marginal effects of CD4-BS2 disruption on CD4-binding on-rates, the second site appears to be essential for stabilizing receptor interaction and triggering envelope activation, which in turn leads to coreceptor binding and progression of the fusogenic process.

The identification of a second receptor-binding surface in the HIV-1 envelope impacts vaccine development. The observation that selected neutralizing antibodies to the CD4-BS like VRC03 and VRC06, which preferentially recognize the envelope trimer, establish contact with CD4-BS2 provides evidence that this region is immunogenic in vivo and, therefore, should be considered as a template for the design of novel vaccine immunogens. It is remarkable that CD4-BS2 is totally disassembled and concealed in monomeric gpl20, which may represent an astute mechanism of HIV- 1 immune evasion. Stabilization of the flexible structure of CD4-BS2 may lead to the design of more effective immunogens capable of inducing potent and broadly reactive neutralizing antibodies against the full quaternary surface that interacts with CD4. Methods

Docking and molecular dynamics. Docking of sCD4 onto the HIV-1 BG505-SOSIP.664 trimer (PDB ID: 4TVP) (Pancera et al., Nature, 514, 455-461, 2014) was obtained by superimposing the crystal structure of a sCD4-bound monomeric gpl20 inclusive of the N-terminal region (PDB ID: 3JWD) (Pancera et al., PNAS, 107, 1166-1171, 2010) to one gpl20 protomer of the trimer; monomeric gpl20 was then hidden. For MD simulation, three sCD4 molecules were added to the BG505-SOSIP.664 trimer. An all atom, isobaric-isothermal (1 atm, 310 K) MD simulation was performed with periodic boundary conditions using the NAMD program (v.2.8) (Phillips et al., J. Comp. Chem., 26, 1781-1802, 2005) on the Biowulf Linux cluster at the NIH, Bethesda, MD (biowulf.nih.gov) after explicitly solvating and energy minimizing the complex followed by warming to 310 K in 10 K increments. Electrostatic interactions were calculated using the Particle-Mesh Ewald summation. The CHARMM force field was used with CHARMM atom types and charges (Brooks et al., J. Comp. Chem., 30, 1545-1615, 2009). For all simulations, a 2 fsec integration time step was used along with a 12 A cutoff. Langevin dynamics were used to maintain temperature at 310 K and a modified Nose-Hoover Langevin piston was used to control pressure.

Surface area and distance measurements. CD4 residues within 3 A of a gp 120 molecule were identified using SYBYL 7.3 (Tripos); the SYBYL MOLCAD program was used to calculate an accessible surface encompassing these residues as well as area. Distances between residues were determined using the VMD program (Humphrey et al, J. Mol. Graph., 14, 33-38, 1996).

Mutagenesis, protein expression and purification. Site-directed mutagenesis was carried out using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). The BG505-SOSIP.664 trimer was expressed by co-transfecting the relevant plasmid (a kind gift of John P. Moore) with a plasmid expressing the cellular protease furin into human embryonic kidney (HEK) 293 free-style (FS) cells. Cell- free supernatants were harvested after 7 days, passed through a 0.22 μηι filter, and loaded onto a Galanthus nivalis (GNA) lectin column (Vector laboratories). After repeated washing with PBS, bound proteins were eluted with 1 M methyl a-D-mannopyranoside in PBS, followed by dialysis toward PBS. Dialyzed samples were concentrated to 2 ml using Amicon Ultra- 15 centrifugal filter units (MWCO 50,000, Millipore) and applied to a PBS pre-equilibrated Superdex 200 column. The peak corresponding to the trimeric form was collected and concentrated, followed by a second round of Superdex 200 purification. Finally, purified trimers were concentrated to 0.5-1 mg/ml and stored at -80 °C. Monomeric HIV- 1 BG505-T332N gpl20 (plasmid donated by Marie Pancera and Peter Kwong) and BaL gpl20 (gene synthesized by Gene Art) were expressed in HEK 293FS. After 7 days, the supernatants were harvested and passed through a 0.22 μηι filter. Purification of gpl20 was carried out by loading filtered supernatants onto a lectin column as described above. The eluted proteins were concentrated using Ami con Ultra- 15 centrifugal filter units (MWCO 10,000, Millipore) and applied to a Superdex 200 column.

Expression of full-length HIV-1 gpl60 or CD4. The full-length CD4 gene (a kind gift of Edward Berger) and the gpl60 gene from BG505-T332N (a gift of Marie Pancera and Peter Kwong) and BaL (a gift of Edward Berger) were mutagenized as described above for the SOSIP trimer. HEK 293T cells were used to express CD4 or gpl60 on the cell-surface membrane. The cells were seeded into 6-well plates at 300,000 cells/well in 2 ml of DMEM containing 10% fetal bovine serum (DMEM 10%). After overnight incubation at 37 °C in humidified atmosphere with 5% CO2, the culture medium was replaced with 1.5 ml of fresh 10% DMEM. DNA-FuGENE 6 complex was prepared by mixing 2 μg DNA with 10 μΐ FuGENE 6 Transfection Reagent (Promega) in Opti-MEM (Gibco) to a final volume of 150 μΐ and incubated for 15 min at room temperature (RT). The cells were transfected by adding the DNA- FuGENE 6 complex dropwise to each well and harvested after 48-60 hours. The same protocol was used for CD4 expression on the surface membrane of Cf2Th/syn-CCR5 cells.

Flow cytometry. HEK 293T cells expressing CD4 or gpl60 were harvested by mechanical shaking and pipetting, washed with PBS and incubated with anti-CD4 or anti-gpl20 antibodies (5 μg/ml) at RT for 30 min. After washing with PBS twice, the cells were incubated with PE-conjugated sheep anti-mouse IgG (Sigma) or goat anti-human IgG (Southern Biotech) at RT for 15 min. The cells were then washed once with PBS, fixed in 2% paraformaldehyde (PFA) and analyzed on a BD FACSCanto™ (BD Biosciences). Data analysis was performed using the FlowJo software.

The human CD4⁺ T-cell clone PM1 (Lusso et al, J. Virol., 69: 3712-3720, 1995) was used to study binding of BG505-SOSIP.664 trimers to native CD4 expressed on the cellular membrane. Purified trimers at 10 μg/ml were pre-incubated with or without 5 μg/ml of 2-domain sCD4 (AIDS Reagent Program) for 10 min at RT. The cells were washed with PBS and incubated with the trimers for 30 min at RT in the presence or absence of sCD4 to prove specificity. After washing twice with PBS, mAb 2G12 was added to the cells at 2 μg/ml for 1 hour at 4 °C, followed by washing. Flow cytometry was performed as described above.

Pseudovirus preparation and infectivity assay. Viral pseudoparticles expressing WT or mutated gpl60 from HIV-1 BG505-T332N or BaL were produced in HEK 293T cells by co-transfecting Env- expressing plasmids together with a backbone plasmid, pSG3^Amv, expressing a full-length HIV-1 clone with a defective Env gene. To produce the pseudoparticles, 2 μg of each Env-expressing plasmid and 4 μg of the backbone plasmid were mixed in Opti-MEM medium (Gibco), and 24 μΐ of FuGENE 6 Transfection

Reagent was added, followed by 15-min incubation at RT. DNA-FuGENE 6 complex was then added to the cells and incubated overnight at 37 °C. After replacing the culture medium with 2 ml fresh 10% DMEM, the cells were incubated for 48 hours and the supernatants containing the pseudoviruses were harvested by centrifugation. For pseudovirus infection, TZM-bl cells (AIDS Reagent Program) were harvested and seeded into 96-well flat bottom plates at 10,000 cells/well in 100 μΐ 10% DMEM. After 30 min at 37 °C, the pseudovirus stock (normalized to 1 ng of p24c_AC protein per mutant) was added to the cells in a total volume of 200 μΐ/well. Reporter gene activation was detected 2 days later using the Luciferase Assay kit (Promega) with a luminometer (PerkinElmer). Relative Light Units (RLU) were recorded, and the final values were normalized against the values obtained with the WT envelope set at 100%. All the samples were tested in duplicate wells.

Luciferase-expressing pseudovirus production and infectivity assay. Pseudoviruses expressing firefly luciferase were produced by co-transfecting Env-expressing plasmids (WT BaL or BG505-T332N) together with a backbone plasmid, pNL4-3.Luc.R^~E^~ (AIDS Reagent Program), at a ratio of 1: 1.

Supernatants containing pseudoviurses were harvested by centrifugation after 48 hours of incubation.

Target cells were prepared by transfecting WT or mutated CD4-expressing plasmids to Cf2Th/syn-CCR5 cells. After incubation for 24 hours, the cells were harvested and seeded to 96-well flat-bottom plates at 10,000 cells/well in 100 μΐ 10% DMEM. Pseudovirus stocks were added to the cells to a total volume of 200 μΐ/well. The reporter gene activity was detected 2 days later using Luciferase Assay kit (Promega) and expressed as relative Light Units (RLU). Serial dilutions of the WT CD4 plasmid were used to generate a reference curve which allowed to extrapolate accurate infectivity values for each mutant based on expression levels, as measured by flow cytometry using mAb OKT4. All samples were performed in duplicate.

ELISA. All the antibodies and envelope proteins used in ELISA experiments were diluted in 0.02% casein in PBS. All samples were washed 3 times after each step with lx wash buffer (R&D Systems). For assessing sCD4 binding to the BG505 SOSIP.664 trimer or monomeric gpl20 (BG505-T332N or BaL), 96- well ELISA plates (Corning) were coated with 2 μg/ml of mAb 2G12 at 4 °C overnight. After blocking with 0.2% casein in PBS, purified trimers (1-3 μg/ml), monomeric BG505-T332N gpl20s (2 μg/ml) or titrated supernatants containing monomeric BaL-gpl20 were added and incubated for 2 hours at RT. After washing, serially diluted 4-domain sCD4 (R&D Systems) was added and incubated for 1 hour at RT. After washing, the samples were incubated with biotin-labeled mAb OKT4 (eBioscience) for 1 hour; after additional washing, horseradish peroxidase (HRP) -conjugated streptavidin (R&D Systems) was added for 1 hour at RT. The reaction was revealed by incubation with the appropriate substrate for 10 min before addition of the stop solution. Light absorption was measured at a wavelength of 450 nm. All samples were tested in duplicate or triplicate wells. Values were normalized toward the respective amount of captured trimeric or monomeric proteins as revealed by either an anti-His tag rabbit antiserum (GenScript; for trimers) or an anti- gpl20 polyclonal goat antibody (anti-SF2, AIDS Reagent Program; for monomers). To evaluate antibody binding, ELISA plates were coated with 5 μg/ml of an anti-gpl20 C-terminus hyperimmune sheep antiserum (D7324) for monomeric gpl20 or with 0.5 μg/ml of rabbit anti-His tag antiserum for trimers. Monomeric gpl20s or trimers were captured on the plates, and various anti-gpl20 antibodies were added to the wells (0.5-5 μg/ml) and incubated for 1 hour, followed by 1-hour incubation with HRP-conjugated goat anti- human IgG antibody (Sigma) at RT. To test binding of mAb 48d, captured trimers were treated with or without 5 μg/ml sCD4 for 30min at RT before adding mAb. The subsequent steps were the same as described above. Values were normalized and shown as relative binding compared to the reference mAb 2G12. gpl20 shedding assay. WT or mutated full-length gpl60 (BaL or BG505-T332N) was expressed on the cell surface of HEK 293T cells by transfection with FuGENE 6 Transfection Reagent. Culture supernatants were collected on day 2 and 4, and then tested for the presence of shed gpl20 by ELISA. Briefly, mAb 2G12 was coated at 2 μg/ml on the plates for capturing soluble gpl20; the anti-SF2 polyclonal antiserum was used to detect captured gpl20. Transfected cells were harvested and stained with 2F5, PG9 and 2G12 antibodies at 5 μg/ml to control for gpl60 expression levels.

SPR analyses. Single-cycle kinetics analysis of 2-domain sCD4 binding to the BG505 SOSIP.664 WT trimer and mutants was performed at 20 °C on a Biacore T200 (GE Healthcare) with HBS-EP (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% surfactant P-20, GE Healthcare). MAb 2G12 was immobilized onto four flow cells of a CM5 sensor to -2,000 response units (RU) using the amine-coupling method. Purified BG505 SOSIP.664 trimers were injected to two sample flow cells at a flow rate of 5 μΐ/min for two minutes, and captured to 300-400 RU. The other two flow cells were left blank as reference. Series dilutions of sCD4 (180 nM, 90 nM, 45 nM, 22.5 nM, 11.25 nM) were injected to both the reference and sample flow cells at 50 μΐ/min in a single cycle, starting from the lowest concentration. Ten minutes of dissociation phase was allowed after the last sCD4 injection. The same injections were carried out using HBS-EP buffer in order to obtain a reference curve. Data were fitted to 1 : 1 Langmuir model with Biacore T200 evaluation software.

SPR analysis of VRC03 and VRC06 binding to WT and mutated BG505-SOSIP.664 trimers was carried out on a Biacore 3000 instrument (GE Healthcare) with HBS-ET (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% Tween 20) buffer. Anti-His tag antibody (His Capture Kit, GE Healthcare) was immobilized onto two flow cells of a CM5 sensor chip (GE Healthcare) to -13000 RU using the amine- coupling method. Each purified trimer at 20 μg/ml was injected into one flow cell to 400-500 RU at 5 μΐ/min flow rate, while the other flow cell served as a control. Then, VRC03 (25 μg/ml) or VRC06 (50 μg/ml) was injected to both flow cells at 25 μΐ/min, followed by a 2-min dissociation phase. Flow cells were regenerated by injecting 25 μΐ of 10 mM glycine pH 1.5 (His Capture Kit, GE Healthcare) twice at a flow rate of 50 μΐ/min. Reference curves were obtained by injection of HBS-ET buffer instead of antibodies, and used to correct the sensorgrams.

CCR5-binding assay. Cf2Th/syn-CCR5 cells (AIDS Reagent Program), which express high levels of CCR5 on their surface membrane, were used to assess binding of soluble BG505-SOSIP.664 trimers to CCR5. The cells were harvested at -80% confluency with enzyme-free cell dissociation buffer (Gibco). His- tagged BG505-SOSIP.664 trimer and mutants were pre-incubated with or without 2-domain sCD4 for 1 hour at 4 °C. After washing with PBS twice, soluble trimers treated with or without sCD4 were incubated with the cells for 1 hour at 4 °C, followed by washing with PBS. PE-conjugated mouse anti-His tag antibody (Miltenyi Biotec) was added to the cells for 1-hour at 4 °C. The cells were washed once with PBS, fixed with 2% PFA and analyzed on a BD FACSCanto instrument. Specificity of binding was assessed by abrogation of the signal with an anti-CCR5 mAb (Becton Dickinson). Data analysis was performed using the FlowJo software. Structure alignments and figures. Structure alignment and figure preparation were performed using PYMOL (The PyMOL Molecular Graphics System, Version 1.7 Schrodinger, LLC). PDB IDs are referenced in individual figures.

Example 2

Immunogenicity of an isolated CD4-BS2 peptide including Residues 54-74 of the BG505 gpl20 sequence.

Rabbits were immunized with the following KLH-conjugated peptide derived from the clade-B HIV-1 isolate BaL: Gly - Cys - Ala - Ser - Asp - Ala - Lys - Ala - Tyr - Asp - Thr - Glu - Val - His - Asn - Val - Trp - Ala - Thr - His - Ala - Cys - Gly - Lys (SEQ ID NO: 30)-KLH. A fast immunization protocol was used based on multiple booster s.c. injections within a 3 months period. The sera obtained from immunized rabbits were tested against the immunizing homologous peptide as well as against recombinant homologous gpl20-BaL or heterologous BG505-SOSIP trimer by ELISA. FIG. 15 shows that immunized rabbits developed significant titers of antibodies against the immunizing peptide, as well as against recombinant gpl20 and, albeit with lower efficiency, against the heterologous SOSIP trimer. These results demonstrate that a peptide containing the main aa. that contribute to the formation of CD4-BS2 is immunogenic in rodents and therefore could be used to induce specific antibodies that may prevent CD4 binding to this region.

Example 3

Immunogenic HIV-1 Env proteins

Elucidation of the complete CD4 binding site (including the CD4-BS1 and CD4-BS2) on the HIV-1 Env trimer allowed for design of recombinant gpl20 proteins and HIV-1 Env ectodomain trimers that include stabilized forms of the complete CD4 binding site (for example, with surface exposure of CD4-BS2 residues that contact CD4, such as gpl20 residues 62, 64, 66 and 207), which can be used to elicit an immune response in vertebrate animals (such as mammals, for example, primates, such as humans) to HIV- 1.

First, HIV-1 Env ectodomain trimers with a neodisulfide bond that stabilizes the complete CD4 binding site were designed. Based on the data provided in example 1, it was determined that a non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113 (HXB2 numbering), such as a disulfide bond between A70C and LI 11C substitutions, should stabilize the CD4 binding site. The non-native disulfide bond holds tryptophan 69 inside a cavity occupied by Trpl 12 and Trp427, to maintain the CD4-BS2 in the prefusion mature closed conformation. A recombinant gpl20 comprising the non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113 or a HIV-1 Env ectodomain trimer including such a recombinant gpl20 should produce an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113.

Second, HIV-1 Env ectodomain trimers with a mutation at tyrosine 61 that stabilizes the complete CD4 binding site were designed. As disclosed in Example 1, mutation of tyrosine 61 (HXB2 numbering) greatly enhances CD4 binding and consequently HIV-1 infectivity. It is believed that tyrosine 61 interferes with formation of the CD4-BS2 in the prefusion mature closed conformation. Based on the data provided in example 1, it was determined that mutation of the Y61 residue, for example by a Y61A or a Y61F substitution, should stabilize the CD4 binding site. The Y61A or Y61F substitution reduces the interference on formation of the CD4-BS2 in the prefusion mature closed conformation when a tyrosine is present at this position. A recombinant gpl20 comprising the Y61A or Y61F substitutions or a HIV-1 Env ectodomain trimer including such a recombinant gpl20 should produce an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the Y61A or Y61F substitution.

Third, HIV- 1 Env ectodomain trimers with a mutation that ablates glycan sequons at positions N262 or N302 were designed. The data provided in Example 1 shows that glycans at these sequons partially shield the surface exposure of CD4-BS2 resides in the gpl20 and HIV-1 Env ectodomain trimer. It is believed that targeted elimination of these glycan sites (for example, by N262Q or S264A substitutions to remove the N262 N-linked glycan sequon, or N301Q or T303A substitution to remove the N301N-linked glycan sequon. A recombinant gpl20 comprising the one or more amino acid substitutions to remove the N- linked glycan sequon at position N262 and/or N301 or a HIV-1 Env ectodomain trimer including the recombinant gpl20 should produce an enhanced immune response to gpl20 compared to a corresponding gpl20 or a HIV-1 Env ectodomain trimer that does not include the one or more amino acid substitutions to remove the N-linked glycan sequon at position N262 and/or N301. Example 4

Immunization of animals

This example describes exemplary procedures for the immunization of animals with the disclosed immunogens, and measurement of the corresponding immune response.

In some examples nucleic acid molecules encoding the disclosed immunogens are cloned into expression vector CMV/R. Expression vectors are then transfected into 293F cells using 293Fectin

(Invitrogen, Carlsbad, CA). Seven days after transfection, cell culture supernatant is harvested and passed over either a 2G12 antibody- or VRCOl antibody-affinity column. After washing with PBS, bound proteins were eluted with 3M MgC , 10 mM Tris pH 8.0. The eluate was concentrated to less than 5 ml with Centricon-70 and applied to a Superdex 200 column, equilibrated in 5 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% azide. The peak corresponding to trimeric HIV-1 Env was identified, pooled, and concentrated or flash-frozen in liquid nitrogen and stored at -80° C. Some proteins are purified using HiTrap IMAC HP Column (GE, Piscataway, NJ), and subsequent gel-filtration using SUPERDEX™ 200 (GE). In some examples the 6x His tag is cleaved off using 3C protease (Novagen, Madison, WI).

For vaccinations with the disclosed immunogens in guinea pigs, 4-6 months old guinea pigs (Strain Hartley) (Charles River Laboratories, MA) are immunized using polylC (High molecular weight, InvivoGen Inc, CA) as the adjuvant. Specifically, four guinea pigs in each group are vaccinated with 25 μg of protein and lOOug of polylC in 400μ1 intramuscularly (both legs, 200μ1 each leg) for example, at week 0, 4, 8, 12, 22.

For vaccinations in non-human primates, Indian origin Rhesus Macaque (bodyweights more than 2kg) are immunized using a disclosed immunogen with polylC-LC as an adjuvant. Specifically, five monkeys in each group are vaccinated with 100 μg of protein and 500 μg polylC-LC in 1ml

intramuscularly in the Quadriceps muscle for example, at week 0, 4, 20.

Sera are collected from the vaccinated animals, for example, at week 2 (Post-1), 6 (Post- 2), 10 (Post- 3), 14 (Post-4) and 24 (Post-5), and subsequently analyzed for their neutralization activities against a panel of HIV- 1 strains, and the profile of antibodies that mediate the neutralization.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such

modifications and variations that fall within the scope and spirit of the claims below.

Claims

It is claimed:

1. An immunogen, comprising:

a recombinant HIV-1 gpl20 comprising one or more amino acid substitutions selected from one of (a) - (d):

(a) a Y61A or a Y61F substitution;

(b) a non-native disulfide bond between cysteine substitutions at one of gpl20 positions 68-72 and one of gpl20 positions 109-113;

(c) mutation of a glycan sequon at position N262 and/or N301; or

(d) a combination of (a) and (b); (a) and (c); (b) and (c); or (a), (b), and (c);

wherein the recombinant gpl20 comprises a CD4 binding site and specifically binds to CD4; and wherein the gpl20 positions correspond to an HIV-1 Env HXB2 reference sequence set forth as SEQ ID NO: 1.

2. The immunogen of claim 1, wherein the non-native disulfide bond comprises a disulfide bond between cysteine residues introduced by A70C and LI 11C substitutions.

3. The immunogen of claim 1 or claim 2, wherein:

the mutation of the glycan sequon at position N262 comprises N262Q and/or S264A substitutions; and/or

the mutation of the glycan sequon at position N301 comprises N301Q and/or T303A substitutions.

4. The immunogen of any one of the prior claims, wherein the recombinant HIV-1 gpl20 comprises or consists of gpl20 positions 31-507, wherein the gpl20 positions correspond to the HIV-1 Env HXB2 reference sequence set forth as SEQ ID NO: 1.

5. The immunogen of any one of claim 1-4, wherein the recombinant gpl20 is a monomer.

6. The immunogen of any one of claims 1-4, comprising a recombinant HIV-1 Env ectodomain trimer comprising three gpl20-gp41 protomers comprising the recombinant gpl20 and a gp41 ectodomain.

7. The immunogen of claim 6, wherein the recombinant HIV-1 Env ectodomain trimer comprises a non-native disulfide bond between cysteine substitutions at one of gpl20 positions 71-75 and one of gp41 positions 553-559, wherein the gpl20 and gp41 positions correspond to the HIV-1 Env HXB2 reference sequence set forth as SEQ ID NO: 1, particularly wherein the non-native disulfide bond comprises a disulfide bond between cysteine residues introduced by A73C and R557C substitutions.

8. The immunogen of claim 6 or claim 7, wherein the recombinant HIV-1 Env ectodomain trimer is stabilized in a prefusion mature closed conformation by:

(a) a non-natural disulfide bond between cysteine substitutions at HIV-1 gpl20 positions 201 and

433;

(b) a non- natural disulfide bond between cysteine substitutions at positions gp41 positions 501 and

605;

(c) a proline substitution at gp41 position 559; or

(d) a combination of (a), (b), and (c); and

wherein the gpl20 and gp41 positions correspond to the HIV-1 Env HXB2 reference sequence set forth as SEQ ID NO: 1.

9. The immunogen of any one of claims 6-8, wherein the gpl20 comprises or consists of HIV- 1 gpl20 positions 31-507 and the gp41 ectodomain comprises or consists of HIV-1 gp41 positions 512-664, and wherein the gpl20 and gp41 positions correspond to the HIV-1 Env HXB2 reference sequence set forth as SEQ ID NO: 1.

10. The immunogen of any one of the prior claims, wherein the gpl20 comprises or consists of an amino acid sequence at least 90% identical to the HIV-1 gpl20 positions 31-507 (HXB2 numbering) of any one of SEQ ID NOs: 1-9, and that comprises the one or more amino acid substitutions.

11. The immunogen of any one of the prior claims, wherein the gpl20 comprises or consists of the amino acid sequence of the HIV-1 gpl20 positions 31-507 (HXB2 numbering) of any one of SEQ ID NOs: 1-9, and further comprises the one or more amino acid substitutions.

12. The immunogen of any one of claims 6-11, wherein

the gpl20 comprises or consists of an amino acid sequence at least 90% identical to the HIV-1 Env positions 31-507 (HXB2 numbering) of any one of SEQ ID NOs: 1-9; and

the gp41 ectodomain comprises or consists of an amino acid sequence at least 90% identical to the HIV-1 Env positions 512-664 (HXB2 numbering) of any one of SEQ ID NOs: 1-9; and

wherein the gpl20 and/or the gp41 ectodomain further comprise the one or more amino acid substitutions.

13. The immunogen of claim 12, wherein

the gpl20 comprises or consists of the amino acid sequence of the HIV-1 Env positions 31-507 (HXB2 numbering) of any one of SEQ ID NOs: 1-9; and

the gp41 ectodomain comprises or consists of the amino acid sequence of the HIV-1 Env positions 512-664 (HXB2 numbering) of any one of SEQ ID NOs: 1-9; and wherein the gpl20 and/or the gp41 ectodomain further comprise the one or more amino acid substitutions.

14. The immunogen of any of the prior claims, wherein the recombinant HIV-1 gpl20 and/or HIV-1 Env ectodomain trimer is soluble in aqueous solution.

15. The immunogen of any of claims 1-13, wherein the recombinant gpl20 and/or HIV-1 Env ectodomain trimer is membrane anchored by a transmembrane domain linked by peptide bond to the C- terminus of each of the gp41 ectodomains.

16. The immunogen of any one of the prior claims, wherein the amino acid substitutions stabilize the CD4 binding site of the recombinant HIV-1 gpl20 and/or the HIV-1 Env ectodomain trimer in a prefusion mature closed conformation.

17. The immunogen of any one of the prior claims, wherein the amino acid substitutions stabilize the CD4 binding site of the recombinant HIV-1 gpl20 and/or the HIV-1 Env ectodomain trimer in a conformation comprising surface exposure of one or more of (such as all of) gpl20 residues 62, 63, 64, 66 and 207 (HXB2 numbering).

18. An isolated peptide comprising or consisting of an amino acid sequence set forth as KAYX₁X₂EVHNV (SEQ ID NO: 17), wherein Xi is aspartate or glutamate and X₂ is threonine, lysine, or arginine; and wherein the peptide is no more than 50 amino acids in length.

19. The isolated peptide of claim 18, comprising or consisting of the amino acid sequence set forth as KAYDTEVHNV (SEQ ID NO: 18).

20. The isolated peptide of claim 18, comprising or consisting of an amino acid sequence set forth as: CASDAKAYX 1X2EVHNVWATHAC, wherein Xi is aspartate or glutamate and X₂ is threonine, lysine, or arginine (SEQ ID NO: 19), and wherein the peptide comprises a disulfide bond between the cysteine residues at positions 1 and 21.

21. The isolated peptide of claim 20, wherein the peptide comprises or consists of the amino acid sequence set forth as CASDAKAYDTEVHNVWATHAC (SEQ ID NO: 20).

22. The isolated peptide of any of claims 18-21, conjugated to a carrier molecule.

23. The isolated peptide of any of claims 18-21, conjugated to a scaffold protein.

24. An isolated nucleic acid molecule encoding the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, a gpl20-gp41 protomer of the recombinant HIV-1 Env ectodomain trimer, or the isolated peptide, of any of the preceding claims.

25. The nucleic acid molecule of claim 24, wherein the nucleic acid molecule encodes a precursor protein of the gpl20-gp41 protomer of the recombinant HIV-1 Env ectodomain trimer.

26. The nucleic acid molecule of claim 24 or claim 25 operably linked to a promoter.

27. An expression vector comprising the nucleic acid molecule of claim 26.

28. The expression vector of claim 27, wherein the expression vector is a viral vector.

29. A pharmaceutical composition for use in eliciting an immune response to HIV-1 in a subject, comprising a therapeutically effective amount of the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, the isolated peptide, the nucleic acid molecule, or the expression vector of any of claims 1-28, and a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, further comprising an adjuvant.

31. A method for eliciting an immune response to HIV-1 in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, the isolated peptide, the nucleic acid molecule, the expression vector, or the pharmaceutical composition of any one of claims 1-30, thereby generating the immune response to HIV-1 in the subject.

32. A method for treating or inhibiting an HIV-1 infection in a subject, comprising administering a therapeutically effective amount of the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, the isolated peptide, the nucleic acid molecule, the expression vector, or the pharmaceutical composition of any one of claims 1-30 to the subject, thereby treating the subject or inhibiting the HIV-1 infection in the subject.

33. The method of claim 31 or claim 32, wherein administering the therapeutically effective amount of the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, the isolated peptide, the nucleic acid molecule, the expression vector, or the pharmaceutical composition to the subject comprises a prime-boost administration.

34. The method of any of claims 30-33, wherein the subject is at risk of or has an HIV-1 infection.

35. Use of the recombinant HIV-1 gpl20, the recombinant HIV-1 Env ectodomain trimer, the isolated peptide, the nucleic acid molecule, the expression vector, or the pharmaceutical composition of any one of claims 1-30 to elicit an immune response to HIV-1 in a subject.