DNA encoded q23-based ENV immunogens and methods of use thereof as an HIV vaccine

Abstract

Disclosed are HIV immunogens. Also disclosed are nucleic acids encoding these immunogens and methods of producing these antigens. Methods for generating an immune response in a subject are also disclosed. In some embodiments, the method is a method for treating or preventing a human immunodeficiency type 1 (HIV-1) infection in a subject.

Description

DNA ENCODED Q23-BASED ENV IMMUNOGENS AND METHODS OF USE THEREOF AS AN HIV VACCINECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 701,459, filed September 30, 2024, which is hereby incorporated by reference herein in its entirety.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under All 66916 and All 65080 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “206193-0145- OOWO_Sequence_Listing.xml,” creation date of September 30, 2025, and having a size of 688,155 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] The HIV envelope glycoprotein (Env), which is the only target for neutralizing antibodies, has several features that make it a challenge for vaccine development. These include a large and dynamic glycan shield, conformational masking of epitopes, and extensive sequence diversity (Stephenson et al., 2020, Annual Review of Immunology 38, 673-703; Wei et al., 2003, Nature 422, 307-312; Kwong et al., 2002, 420, 678-82; Moore et al., 2001, Journal of Virology 75, 5721-5729). As a result of these properties, the antibody response to Env is usually strainspecific (Moore, 2018, Current HIV research 16, 21-28). However, 10-20% of infected individuals develop broadly neutralizing antibodies (bnAbs) targeting conserved epitopes (Gray et al., 2011, Journal of Virology 85, 4828-4840; Doria-Rose et al., 2010, Journal of Virology 84, 1631-1636; Haynes et al., 2012, Nat Biotechnol, 30, 423-433; Kwong et al., 2018, Cell Press, 48, 855-871; Landais et al., 2016, PLOS Pathogens 12, el005369; Hraber et al., 2014, AIDS(London, England) 28, 163-169; Rusert et al., 2016, Nature Medicine, 22, 1260-1267), and bnAbs have been shown to be protective against infection by diverse heterologous viruses in humans and rhesus macaques (RMs) (Gautam et al., 2016, Nature 533, 105-109; Corey et al.,2021, New England Journal of Medicine 384, 1003-1014; Julg et al., 2017, Science Translational Medicine 9, eaall321; Hessell et al., 2009, Nature Medicine, 15, 951-954).

[0005] Among the various classes of bnAbs, antibodies targeting the V2 apex are some of the most frequently elicited during infection, are highly potent, and have relatively straightforward developmental pathways (Landais et al., 2017, Immunity, 47, 990-1003. el009; Doria-Rose et al., 2014, Nature, 508, 55-62; Roark et al., 2021, Science, 371, eabd2638). These features make the apex a promising target for vaccine development. However, to date, vaccination has not been able to elicit V2 apex bnAbs in outbred animal models, and there is a need for immunogens capable of doing so. V2 apex bnAbs primarily rely on their long CDRH3s for binding to Env, forming distinct microdomains that contact several glycans and the C-strand of the V1V2 region (Roark et al., 2021, Science, 371, eabd2638; Willis et al., 2022, Immunity 55, 2149-2167. e9; Lee et al., 2017, Immunity 46, 690-702; Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; McLellan et al., 2011, Nature 480, 336-343; Pejchal et al., 2010, Proceedings of the National Academy of Sciences 107, 11483-11488; Julien, et al., 2013, Proceedings of the National Academy of Sciences of the United States of America 110, 4351-6; Gorman et al., 2020, Cell Reports 31, 107488; Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384.)

[0006] However, V2 apex bnAb precursors are estimated to have low frequencies in the naive human B cell repertoire, making priming a major hurdle to their elicitation (Willis et al.,2022, Immunity 55, 2149-2167. e9). This challenge is compounded by the fact that, unlike bnAb precursors to other epitopes, V2 apex bnAbs utilize diverse V, D, and J genes, making it difficult to identify V2 apex bnAb precursors based on sequence features alone (Willis et al., 2022, Immunity 55, 2149-2167.e9).

[0007] Thus, there is a need for immunogens that can be used to elicit an immune response to HIV. This invention fulfills this need.SUMMARY OF THE INVENTION

[0008] In some embodiments, the invention provides an HIV immunogenic composition comprising a polypeptide comprising an amino acid sequence encoding HIV Env.

[0009] In some embodiments, the amino acid sequence comprises SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, or 190. In some embodiments, the amino acid sequence is at least90% identical to SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, or 190. In some embodiments, the amino acid sequence is at least 70% of the length of SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, or 190. In some embodiments, the amino acid sequence is at least 90% identical to and at least 70% the length of SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, or 190.

[0010] In some embodiments, said polypeptide further comprises a nanoparticle scaffold.

[0011] In some embodiments, the amino acid sequence is encoded by SEQ ID NO: 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the amino acid sequence is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the amino acid sequence is encoded by a nucleotide sequence at least 70% of the length of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the aminoacid sequence is encoded by a nucleotide sequence at least 90% identical to and at least 70% the length of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 or SEQ ID NO: 191-288.

[0012] In some embodiments, the invention provides a nucleic acid molecule encoding a variant HIV Env polypeptide.

[0013] In some embodiments, the variant HIV Env polypeptide sequence comprises SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, or 190. In some embodiments, the variant HIVEnv polypeptide sequence is at least 90% identical to SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, or 190. In some embodiments, the variant HIV Env polypeptide sequence is at least 70% of the length of SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, or 190. In some embodiments, the variant HIV Env polypeptide sequence is at least 90% identical to and at least 70% the length of SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, or 190.

[0014] In some embodiments, the nucleotide sequence encoding the variant HIV Env polypeptide comprises SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the nucleotide sequence encoding the variant HIV Env polypeptide comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the nucleotide sequence encoding the variant HIV Env polypeptide comprises a nucleotide sequence at least 70% of thelength of SEQ ID NO: 99, 101, 103, 105, 107, 109, 1 11, 113, 115, 117, 1 19, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, or SEQ ID NO: 191-288. In some embodiments, the nucleotide sequence encoding the variant HIV Env polypeptide comprises a nucleotide sequence at least 90% identical to and at least 70% the length of SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 or SEQ ID NO: 191-288.

[0015] In some embodiments, the invention provides a composition comprising a nucleotide sequence encoding the variant HIV Env polypeptide.

[0016] In some embodiments, the invention provides a method of inducing an immune response against HIV in a subject in need thereof, comprising administering to the subject the polypeptide, nucleic acid molecule or composition described herein.

[0017] In some embodiments, the invention provides a method of treating or preventing infection by HIV in a subject, comprising administering to the subject the composition described herein. In some embodiments, said subject is a human. In some embodiments, the subject is infected with HIV or at risk of becoming infected with HIV.

[0018] In some embodiments, the invention provides a method of treating or preventing a disease or disorder associated with HIV infection in a subject, comprising administering to the subject the composition described herein. In some embodiments, said subject is a human. In some embodiments, the subject is infected with HIV or at risk of becoming infected with HIV. In some embodiments, the disease or disorder associated with HIV infection is AIDS.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1, comprising Figure 1A through Figure IF, depicts the results of example experiments using structural characteristics of axe-like bnAbs to estimate axe-like B cell frequency in the human repertoire. Figure 1A depicts an overview of the study, starting from in silico analysis to immunogen design to in vivo characterization and re-elicitation of Axe Abs in NHPs. Figure IB depicts CHOI -like antibody search results from the OAS database. Each point on the graph represents a donor that had at least one sequence matching the search criteria. All searches are detailed in the table. The motif used was a DH gene motif, Y[YQK]GSG (SEQ IDNO:360, SEQ ID NO:361 and SEQ ID NO:362), but a specific DH gene was not included. Figure 1C depicts the AlphaFold2 model of CHOI has a low CDRH3 CA RMSD to the published CH04 structure (PDB ID 3tcl). Figure ID depicts AlphaFold models from CDRH3 saturated mutagenesis where each position along the CHOI CDHR3 was mutated to every possible residue. The x-axis shows the native CHOI CDRH3 sequence while the y-axis shows the mutations. The minimum CDRH3 CA RMSD (A) to the AlphaFold-generated control structure is shown for each mutation. Small RMSD values (lighter) represent positions where the mutations did not greatly affect the fold while large RMSD values (darker) represent positions where the mutations greatly perturbed the CDRH3 fold. Figure IE depicts a scatter plot showing the structural definition of an axe CDRH3 fold applied to CH04 (PDB ID 3tcl), where darker points show beta-sheet residues that are at least 20 A away from the start of the CDRH3. The pie chart on the left shows the distribution of donors (n=215) that have no structural or sequence axe-like motif, donors that have a sequence axe-like motif but were not predicted to have any axe-like structures, and donors that have a sequence axe-like motif that folded into an axe-like structure. The pie chart on the right is the same, but only shows donors which have at least IM sequences in the OAS database. Figure IF depicts the structures of antibodies found from Search 7, 3 of which are predicted to be axe-like (seql0941, seql5061, seq20621) and 1 was predicted to not be axe-like (seql0821). The plot below the structures shows the ratio of frames within each MD simulation where the structure was classified as axe-like.

[0020] Figure 2, comprising Figure 2A through Figure 2D, depicts the result of OAS data base searches wherein diverse CDRH3 sequences were found. Figure 2A depicts CHOI Search 7 CDRH3 length distribution, the distribution of IGHJ genes, and the distribution of IGHD genes, before redundant sequences were removed. Figure 2B depicts PG9-like antibody search results from the OAS database. Each point represents a donor that had at least one sequence matching the search criteria, as detailed in the table. Figure 2C depicts PG9 Search 7 CDRH3 length distribution, the distribution of IGHJ genes, and the distribution of IGHD genes, before redundant sequences were removed. Figure 2D depicts results of the rhesus-related OAS searches broken down by CDRH3 length.

[0021] Figure 3, comprising Figure 3A and Figure 3B depicts axe-like structural classification. Figure 3A depicts the structural definition of an axe-like CDRH3 applied to PG9 (PDB ID: 5VJ6), 41328-a, and V033-a, with each respective CDRH3 shown above each scatterplot, as well as a structure of their variable heavy (dark) and light chains (light) shown as a surface representation with the CDRH3 pulled out. Figure 3B depicts CDRH3 residue classifications (scatter plots) and CDRH3 structures for the antibodies classified as axe-like from the SAbDab database (excluding CHOI -lineage structures, the second CAP256-VRC26 PDB (40RG), and the second 10E8 PDB (5JNY)).

[0022] Figure 4, comprising Figure 4A through Figure 4F, depicts experimental results estimating the frequency rhesus-like axe antibodies within the human repertoire. Figure 4A depicts various rhesus-like antibody search results from the OAS database, broken down into CDRH3 lengths (all lengths, at least 20 AA in length, and at least 23 AA in length). Each point on the graph represents a donor that had at least one sequence matching the search criteria. The three searches are detailed in the table. Figure 4B depicts sequence alignments of the two rhesus antibodies with rhesus and human genes, and the sequences found in Search 1. Figure 4C depicts the enrichment of IGHD genes within Search 2, with positive enrichment in blue and negative enrichment in red. Figure 4D depicts structures of antibodies found in Search 3, 3 of which are classified as axe-like and 1 is not classified as axe-like. Figure 4E depicts the ratio of frames within each trajectory where the structure was classified as axe-like. Figure 4F depicts the distribution of highly sequenced donors (1M+ sequences, n=37) amongst the most expanded searches of CHOI -like, rhesus-like, and PG9-like antibodies.

[0023] Figure 5, comprising Figure 5A through Figure 5E, depicts the results of characterization and DNA-delivery of Q23-based immunogens. Figure 5A depicts the antigenic profile of Q23.MD39 and Q23.gpl20.foldon against a panel of broadly neutralizing and nonneutralizing antibodies. Figure 5B depicts model and NS-EM 2D class averages of Q23. Ferritin nanoparticles. Figure 5C depicts ELISA area under the curve of binding of Q23.gpl20.foldon, Q23.MD39, and Q23. Ferritin to axe-like V2 apex bnAb iGLs. Figure 5D depicts a time course of vaccine-specific serum -IgG binding endpoint titers in WT BALB / c mice immunized with 25 pg of DNA-delivered Q23.MD39. Circles indicate individual mice, and vertical dotted lines indicate immunization timepoints. Figure 5E left depicts an overview of the ACTIV assay, and Figure 5E right depicts ELISA area under the curve binding of muscle lysate from mice immunized with Q23.MD39 or BG505.MD39 to various antibodies.

[0024] Figure 6, comprising Figure 6A through Figure 6C, depicts the characterization of Q23-based immunogens. Figure 6A depicts size-exclusion chromatography trace of Q23.MD39.Figure 6B depicts size exclusion chromatography with multi -angle light scattering trace of Q23.MD39. Lower line indicates protein molecular weight of the main peak. Figure 6C depicts size-exclusion chromatography trace of Q23.Ferritin. Figure 6D depicts antigenic profile of Q23. Ferritin against a panel of broadly neutralizing and non-neutralizing antibodies.

[0025] Figure 7, comprising, Figure 4A and Figure 4B, depict immunogenicity of DNA- delivered Q23 immunogens.

[0026] Figure 8, comprising Figure 8A through Figure 8D, depict Cryo-EM structure of Q23.MD39 in complex with the CHOI iGL. Figure 8 A depicts density maps of Q23.MD39 / 35O22, Q23.MD39 / CH01 iGL / 35022. Figure 8B depicts overlayed atomic models of CHOI iGL, CH03 (PDB: 5ESV), and CH04 (PDB: 5ESZ). Figure 8C depicts interactions at the epitope-paratope interface of CHOI iGL and Q23.MD39. Figure 8D depicts a comparison of N160 glycan on gpl20B of Q23.MD39 with N139 glycan in the atomic models of CH03 and CH04.

[0027] Figure 9, comprising Figure 9A through Figure 9D, depict a single-round mammalian display approach for the identification of V033 iGL affinity enhancing mutations. Figure 9A depicts an overview of the mammalian display approach. An immunogen variant library is packaged into lentiviral vectors, following which 293t cells are transduced with the library. Cells displaying the immunogen variant library are then stained with an antibody of interest, sorted, and deep sequenced to identify enriched mutations. Figure 9B depicts positive and negative enrichments following a single round of sorting against the V033 iGL. Black dots indicate the WT residue at each position. Boxes indicate mutations evaluated in Figure 9C. Figure 9C depicts ELISA area under the curve binding values of the V033 iGL to Q23.MD39 site-directed mutants incorporating mammalian-display identified mutations. AUC values are normalized to WT. Figure 9D depicts ELISA area under the curve binding values of the VO33 iGL to Q23.MD39 mutants sequentially incorporating mammalian-display identified mutations. AUC values are normalized to WT.

[0028] Figure 10 depicts SPR binding affinities of Q23.MD39, Q23.V033GT, and Q23.RH-GT to axe-like bnAb iGLs.

[0029] Figure 11, comprising Figure 11A through Figure 1 IE, depicts infectivity, replication kinetics, and evolution of SHIV-Q23.V033GT. Figure 11A depicts TZM-bl and p27 antigen ELISA titers of WT SHIV-Q23.17 and SHIV-Q23.V033GT. Figure 1 IB depicts viralreplication kinetics in SHIV-Q23.V033GT infected macaques. Macaque CL60 was euthanized 32 weeks post-infection due to symptoms associated with rapid progression to AIDS. Figures 11C through 1 IE depict the results of single genome sequencing of the V1V2 region (HXB2 residues 130-199) of circulating plasma virus in macaques CL60 (Figure 11C), CK52 (Figure1 ID), and CK11 (Figure 1 IE). Mismatches to the transmitted / founder SHIV are highlighted. N = number of viruses. Numbers indicate HXB2 residue numbers, and timepoints of virus isolation are labelled on the left of the topmost sequence.

[0030] Figure 12, comprising Figure 12A through Figure 12H, depicts the results of example experiments demonstrating infection with a germline-targeting SHIV elicits axe-like antibodies to the V2 apex. Figure 12A depicts neutralization IC50 values of SHIV-Q23.17 and SHIV-V033GT against a panel of non-neutralizing antibodies, the mature V033-a.01 bnAb, and the V033 iGL. Figure 12B depicts longitudinal plasma ID50 titers against MLV, autologous mutants, and tier 1 A viruses. MLV, Murine Leikemia Virus. Figure 12C depicts single genome sequencing of circulating plasma viruses at week 12 post-infection in rhesus macaques infected with SHIV-BG505, SHIV-Q23.17, and SHIV Q23.V033GT. C-strand residues that are the primary site of Env escape (166-173) from V2 apex bnAbs are highlighted in teal. V2b residues that are potential sites of glycan additions are highlighted in blue. N = number of viruses. Figure 12D depicts hamming distance of V2 residues C131-C198 at week 12 in the RMs from Figure 12C. Error bars indicate a 95% confidence interval. Figure 12E depicts neutralization ID50 curves of week 28 plasma from macaques CK11 and CK52 against a panel of heterologous Tier2 viruses. Figure 12F depicts 3D reconstruction of selected NS-EM 3D classes that displayed V2 apex binding antibodies for macaques CK11 and CK52 at week 24. Figure 12G depicts 3D reconstruction of a NS-EM V2 apex binding class from CK52 at week 24, docked with atomic models of BG505_DS / 41328, BG5O5_DS / VO33, BG505_DS / PG9, and Q23.MD39 / CH01 iGL. Figure 12F depicts Cryo-EMPEM reconstructed density map of a V2 apex antibody binding to Q23.V033GT.

[0031] Figure 13, comprising Figure 13A through Figure 13C, depicts the generation of a pan-axe targeting immunogen. Figure 13A depicts an overview of the approach for immunogen design. Mutations enriched in multiple mammalian display mutagenesis sorts against a structural iGL class are identified and incorporated into an immunogen. Figure 13B depicts ELISA area under the curve binding of Q23.MD39, Q23.V033GT, and Q23.RH-GT against the axe-like V2apex bnAb precursors. Figure 13C left depicts example experiments wherein HuD3-3 / J6 mice were primed and boosted with 25pg of DNA-encoded Q23.RH-GT adjuvanted with 0.5pg of IL- 2 plasmid. Figure 13C right depicts ELISA binding of serum from mice immunized with Q23.RH-GT or WT Q23. Ferritin to Q23.RH-GT or a Q23 epitope knockout. Shapes indicate individual mice. Solid lines indicate binding to Q23.RH-GT. Dashed lines indicate binding to Q23.KO.

[0032] Figure 14 depicts amino acid sequence alignment of Env Q23.17 and Q23.17- based constructs. Mismatches to Q23.MD39 are highlighted. Numbers indicate HXB2 residue. Env Q23.17 (SEQ ID NO:309), Q23.MD39 (SEQ ID NO:310), Q23.KO (SEQ ID NO:311), Q23.V033GT (SEQ ID NO:312), Q23.RH-GT (SEQ ID NO:313), Q.23 Ferritin (SEQ ID NO:314), Q23.PDGFR (SEQ ID NO:315).

[0033] Figure 15, comprising Figure 15A and Figure 15B, depicts the CryoEM data processing pipeline for Q23.MD39 / CH01 iGL / 35022 dataset as well as CryoEMPEM dataset for CK52. Figure 15A depicts the CryoEM data processing pipeline with a representative raw micrograph. Figure 15B depicts the CryoEMPEM data processing pipeline of CK52 polyclonal response at week 24 in CryoSPARC.

[0034] Figure 16 depicts CryoEM of Q23 in complex with CHOI UCA.

[0035] Figure 17 depicts Q23 mutants by computational design.

[0036] Figure 18 depicts PTs-ELISA binding of Q23 CD4bs mutants.

[0037] Figure 19 depicts the mammalian display strategy.

[0038] Figure 20 depicts NNK scanning region of site-saturation library.

[0039] Figure 21 depicts the results of sorting against CD4bs and bnAbs and their intermediates.

[0040] Figure 22 depicts the conformation of mutations derived from mammalian display.

[0041] Figure 23 depicts the results of directed evolution by mammalian display to guide Q23-based immunogen designs for VRC01 -class early intermediates. Selected GT8 elicited antibodies from trail I are shown.

[0042] Figure 24 depicts strong binding mutants against GT8 elicited antibodies from trail I.

[0043] Figure 25 depicts combination mutant enhanced binding to early intermediates ofVRC01 -Class.

[0044] Figure 26, comprising Figure 26A and Figure 26B, depicts NS-EMPEM of the polyclonal antibody responses to Q23.V033GT in rhesus macaques CK11 and CK52. Figure 26A depicts a representative raw micrograph, 2D classes, and 3D reconstructions of Fab / Q23.V033GT complexes from CK11 at week 24 post-infection. Figure 26B depicts a representative raw micrograph, 2D classes, and 3D reconstructions of Fab / Q23.V033GT complexes from CK52 at week 24 post-infection.DETAILED DESCRIPTION

[0045] The invention is based on the development of an immunogen that recapitulates the antigenicity of an HIV envelope protein (Q23.17). In some embodiments, the immunogen is suitable for vaccination strategies to stimulate an immune response (e.g., HIV immune response) in a subject. In one embodiment, the immunogen is able to elicit detectable antibody responses and heterologous neutralizing antibodies against HIV-1. In some embodiment, the immunogen of the invention elicits neutralizing antibodies with axe-like properties.

[0046] In one embodiment, the composition comprises a peptide comprising an amino acid sequence capable of expressing one or more HIV antigens in the subject and a pharmaceutically acceptable excipient.

[0047] In one embodiment, the composition comprises nucleotide sequences capable of expressing an HIV antigen in the subject and a pharmaceutically acceptable excipient. In one embodiment, the nucleic acid molecule comprises a promoter operably linked to a coding sequence that encodes an HIV antigen. In some embodiments, the composition comprises nucleotide sequences capable of expressing a self-assembling ferritin nanoparticle decorated with an HIV antigen.Definitions

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications,patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0049] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0050] “Adjuvant” as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen.

[0051] “Antibody” as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody can be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0052] “Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.

[0053] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0054] “Complement” or “complementary” as used herein means Watson-Crick (e.g., A- T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0055] “Consensus” or “Consensus Sequence” as used herein may mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis ofan alignment of multiple subtypes of a particular antigen. The sequence may be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens, such as fusion proteins, may be manipulated to generate consensus sequences (or consensus antigens).

[0056] “Endogenous antibody” as used herein may refer to an antibody that is generated in a subject that is administered an effective dose of an antigen for induction of a humoral immune response.

[0057] “Fragment” as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below.

[0058] “Fragment” or “immunogenic fragment” with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross reacts with a full-length wild type strain HIV antigen. Fragments of consensus proteins can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of a consensus protein.

[0059] As used herein, the term “genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a codingsequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

[0060] “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0061] “Immunogenic polypeptide” refers to a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogenic polypeptide can lead to protective immunity against a pathogen of interest.

[0062] “Immune response” as used herein means the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0063] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0064] Nucleic acids can be single stranded or double stranded, or can contain portions ofboth double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0065] “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0066] A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0067] “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[0068] “Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a modified HIV envelope immunogens set forth herein. Signal peptides / leader sequences typically direct localization of aprotein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0069] “Subject” as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0070] “Substantially identical” as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

[0071] “Treatment” or “treating,” as used herein can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to an animal after clinical appearance of the disease.

[0072] “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0073] Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0074] A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0075] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a selfreplicating extrachromosomal vector, and preferably, is a DNA plasmid.

[0076] “Polypeptide” is used in its conventional meaning, i.e. , as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, polypeptides, and proteins are included within the definition of polypeptide, and such terms can be used interchangeably herein unless specifically indicated otherwise. This term also includes post expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like, as well as other modifications known in the art, both naturally occurring and non- naturally occurring. A polypeptide can be an entire protein or a subsequence thereof. A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the disclosure and evaluating one or more biological activities of the polypeptide as described herein and / or using any of some techniques well known in the art.

[0077] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (for example, antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, accordingly, its underlying DNA coding sequence, whereby a protein with like properties is obtained. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.

[0078] Variant sequences include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of this disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiaryprotein structure. Such conservative modifications include amino acid substitutions, additions, and deletions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, pro line, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0079] “Sequence identity” or“homology” refers to the percentage of residues in the polynucleotide or polypeptide sequence variant that are identical to the non- variant sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. In particular embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.

[0080] Polypeptide variant sequences may share 70% or more (i.e. 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences recited in this disclosure. Polypeptide variants may also include polypeptide fragments comprising various lengths of contiguous stretches of amino acid sequences disclosed herein. Polypeptide variant sequences include at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, or more contiguous peptides of one or more of the sequences disclosed herein as well as all intermediate lengths therebetween.

[0081] The terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen but not to other antigens. Typically, the antibody binds with an equilibrium dissociation constant (KD) of approximately less than 10 6 M, such as approximately less than 10 7 M, 10 8 M, 10 9 M or 10 10 M or even lower when determined by, e.g., ELISA, equilibrium dialysis or surface plasmon resonance (SPR) technology in a BIACORE® 2000 surface plasmon resonance instrument using the predetermined antigen, e.g., an epitope on the viral envelope of HIV-1 , e.g., gpl20, as the analyte and the antibody as the ligand, or Scatchard analysis of binding of the antibody to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinitythat is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g. , BSA, casein) other than the predetermined antigen or a closely-related antigen.

[0082] In the context of the present invention, a “truncated” envelope protein is one which contains less than a full-length cytoplasmic domain, but retains surface antigenic determinants against which an immune response is generated, preferably a protective immune response, and it retains sufficient envelope sequence for proper precursor processing and membrane insertion. The skilled artisan can produce truncated virus envelope proteins using recombinant DNA technology and virus coding sequences, which are readily available to the public. For example, the coding sequence of a virus envelope protein can be engineered for expression in a baculovirus expression vector, for example, using a commercially available baculovirus vector, under the regulatory control of a virus promoter, with appropriate modifications of the sequence to allow functional linkage of the coding sequence to the regulatory sequence, and truncation (deletion) of the portion of the coding sequence which encodes the cytoplasmic domain of the envelope protein, again with appropriate translation stop signals and sequences which allow operable splicing of the truncated envelope and associated sequences into the vector.

[0083] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.Description:

[0084] The invention is based on the development of an immunogen that recapitulates the antigenicity of an HIV envelope protein (Q23.17). In some embodiments, the immunogen is suitable for vaccination strategies to stimulate an immune response (e.g., HIV immune response) in a subject. In one embodiment, the immunogen is able to elicit detectable antibody responses and heterologous neutralizing antibodies against HIV-1. In some embodiment, the immunogen of the invention elicits neutralizing antibodies with axe-like properties.

[0085] In one embodiment, the composition comprises a peptide comprising an amino acid sequence capable of expressing one or more HIV antigens in the subject and a pharmaceutically acceptable excipient.

[0086] In one embodiment, the composition comprises nucleotide sequences capable of expressing an HIV antigen in the subject and a pharmaceutically acceptable excipient. In one embodiment, the nucleic acid molecule comprises a promoter operably linked to a coding sequence that encodes an HIV antigen. In some embodiments, the composition comprises nucleotide sequences capable of expressing a self-assembling ferritin nanoparticle decorated with an HIV antigen.

[0087] In one embodiment, the immunogen comprises a polypeptide having a sequence that is at least 75% identical to SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, or 190. In one embodiment, the immunogen comprises a polypeptide having a sequence that is at least 75% identical to SEQ ID NO: 92. In one embodiment, the immunogen comprises a polypeptide having a sequence that is at least 75% identical to SEQ ID NO: 190.

[0088] The above-described immunogens may elicit bNAbs. bNAbs are neutralizing antibodies that neutralize multiple HIV-1 viral strains. bNAbs are unique in that they target conserved epitopes of the virus. Examples of broadly neutralizing antibodies may include, without limitation, VRC26.25, PCT64-24E, VRC38.01, PG9, PGDM1400, CHOI, BG18, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, VRC29.03, PGT121, 10-1074, N49-P7, N6, NC-Cowl, I0MA, CH235, CH235.12, bl2, VRC01, 3BNC117, CH103, VRC-PG05, VRC34.01, ACS202, PGT151, 35022, 8ANC195, DH511.11P. Among these bNAbs, BG18, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, VRC29.03, PGT121, 10-1074 broadly neutralizing antibodies bind specifically to V3 glycans.

[0089] In another aspect, this disclosure provides immunogen polypeptides that are multimerized. However, the invention is not limited to any means of multimerizing. Rather, the invention includes any means of multimerizing including but is not limited to multimerizing on a nanoparticle, on a virus-like particle (VLP) (e g., retrovirus-like particle, HIV-like particle), and the likes. Virus-like particles, or retrovirus-like particles, in the context of the present disclosure, are membrane-surrounded structures comprising viral envelope proteins embedded within the membrane of the host cell in which they are produced, and preferably, additional viral core proteins in the VLPs. These VLPs do not contain intact viral nucleic acid, and they are non-infectious. Desirably, there is sufficient envelope protein on the surface of the VLP so that when a VLP preparation is formulated into an immunogenic composition and administered to an animal or human, an immune response (cell-mediated or humoral) is raised.

[0090] In another aspect, this disclosure provides a protein complex comprising at least one above-described immunogen polypeptide multimerized via covalent or non-covalent bonding / interaction (e.g., van der Waals interactions). For example, two or more immunogen polypeptides may be cross-linked by one or more cross-linkers. Crosslinkers are reagents having reactive ends to specific functional groups (e.g. , primary amines or sulfhydryls) on proteins or other molecules. Crosslinkers are capable of joining two or more molecules by a covalent bond. Crosslinkers include but are not limited to amine- to-amine crosslinkers (e.g., disuccinimidyl suberate(DSS)), amine-to-sulfhydryl crosslinkers (e.g. , N-g-maleimidobutyryl- oxysuccinimide ester (GMBS)), carboxyl-to-amine crosslinkers (e.g., dicyclohexylcarbodiimide (DCC)), sulfhydryl-to-carbohydrate crosslinkers (e.g., N-b- maleimidopropionic acid hydrazide (BMPH)), sulfhydryl-to-sulfhydryl crosslinkers (e.g., 1,4- bismaieimidobutane (BMB)), photoreactive crosslinkers (e.g. , N-5-azido-2- nitrobenzoyloxysuccinimide (ANB-NOS)), chemo selective ligation crosslinkers (e.g., NHS- PEG4-Azide).

[0091] In one embodiment, the present invention provides an immunogenic composition comprising one or more nucleic acid molecules that are capable of generating in a mammal an immune response against an HIV antigen. The present invention also provides isolated nucleic acid molecules that are capable of generating in a mammal an immune response against an HIV antigen. In one embodiment, the immunogenic composition comprises amino acid sequences for an HIV antigen having the amino acid sequence set forth in SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, or 190 or fragments or variants thereof. In one embodiment, the immunogenic composition comprises amino acid sequences for an HIV antigen having the amino acid sequence set forth in SEQ ID NO:92 or a fragment or variant thereof. In one embodiment, the immunogenic composition comprises amino acid sequences for an HIV antigen having the amino acid sequence set forth in SEQ ID NO: 190 or a fragment or variant thereof.

[0092] In one embodiment, the invention provides compositions comprising a nucleic acid molecule comprising a nucleotide sequence that encodes an HIV antigen. In oneembodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ IDNO:l-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, or 190, or fragments or variants thereof. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 92 or a fragment or variant thereof. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO: 190 or a fragment or variant thereof. In one embodiment, a nucleotide sequence which encodes an HIV antigen is provided as SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, SEQ ID NO: 191-288, or fragments or variants thereof. In one embodiment, a nucleotide sequence which encodes an HIV antigen is provided as SEQ ID NO: 282 or a fragment or variant thereof. In one embodiment, a nucleotide sequence which encodes an HIV antigen is provided as SEQ ID NO: 288 or a fragment or variant thereof.

[0093] Compositions that comprise one or more nucleotide sequence that encode an HIV antigen may be on a single plasmid. In one embodiment, a composition comprises a single plasmid that encodes an HIV antigen under a single promoter.

[0094] In one embodiment, an HIV antigen is operably linked to one or more regulatory elements. In one embodiment, a regulatory element is a leader sequence. In one embodiment, a regulatory element is a start codon. In one embodiment, a regulatory element is at least one stop codon.

[0095] When taken up by a cell, the DNA plasmids can remain in the cell as separate genetic material. Alternatively, RNA may be administered to the cell. It is also contemplated to provide a genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the target protein or the immunomodulating protein. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered. Such genetic constructs may therefore be recombinant nucleic acid molecules.

[0096] The recombinant nucleic acid molecule can include one or more recombinant nucleotide sequence constructs. The recombinant nucleotide sequence construct can include a heterologous nucleotide sequence that encodes a viral antigen, a fragment thereof, a variant thereof, or a combination thereof.

[0097] The recombinant nucleotide sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide. In some embodiments, nucleic acid constructs may be provided in which the coding sequences for the proteins described herein are linked to IgE leader peptide, or such IgE leader is removed. In some embodiments, proteins described herein are linked to IgE signal peptide, or such IgE leader is removed.

[0098] The one or more vectors can be a plasmid. The plasmid may be useful for transfecting cells with the recombinant nucleotide sequence construct. The plasmid may be useful for introducing the recombinant nucleotide sequence construct into the subject. The plasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.

[0099] The plasmid may also comprise a mammalian origin of replication in order to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in a cell. The plasmid may be pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may comprise the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The backbone of the plasmid may be pAV0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

[0100] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E.coli). The plasmid may also be pYES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.Vaccines and Immunogenic Compositions

[0101] Immunogenic compositions, such as vaccines, are provided comprising an optimized consensus sequence, an optimized consensus-encoded antigen, a fragment thereof, a variant thereof, or a combination thereof. The immunogenic composition can significantly induce an immune response of a subject administered with the immunogenic composition against the HIV antigen. The vaccine may comprise a plurality of the nucleic acid molecules, or combinations thereof. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

[0102] The immunogenic composition can be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination vaccine. The vaccine can include an optimized consensus nucleotide sequence encoding an antigen. The nucleotide sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleotide sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the antigen by a peptide bond. The peptide vaccine can include an antigen, a variant thereof, a fragment thereof, or a combination thereof. The combination DNA and peptide vaccine can include the above described optimized consensus nucleotide sequence and the encoded antigen.The vaccine can be a DNA vaccine. DNA vaccines are disclosed in US Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporated herein fully by reference. The DNA vaccine can further comprise elements or reagents that inhibit it from integrating into the chromosome.

[0103] The vaccine can be an RNA of the one or more HIV antigens. The RNA vaccine can be introduced into the cell.

[0104] The vaccine can use recombinant vectors to deliver antigen, subunit vaccines, and glycoprotein vaccines, for example, but not limited, the vaccines described in U.S. Patent Nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5, 110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,3 64; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are each incorporated herein by reference.

[0105] The vaccine of the present invention can have features required of effective vaccines such as being safe so that the vaccine itself does not cause illness or death; beingprotective against illness; inducing protective T cell responses; and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0106] Provided herein is an immunogenic composition capable of generating in a mammal an immune response against HIV. The immunogenic composition may comprise each plasmid as discussed above. The immunogenic composition may comprise a plurality of the plasmids, or combinations thereof. The immunogenic composition may be provided to induce a therapeutic or prophylactic immune response. In one embodiment, the composition of the invention can induce immunization after one immunization.

[0107] Immunogenic compositions may be used to deliver nucleic acid molecules that encode one or more consensus HIV antigen. Immunogenic compositions are preferably compositions comprising plasmids.

[0108] The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide.

[0109] In one embodiment, the nucleic acid molecule comprises an optimized nucleic acid sequence. The optimized sequence can comprise a consensus sequence and / or modification(s) for improved expression. Modification can include codon optimization, RNA optimization, addition of a kozak sequence for increased translation initiation, and / or the addition of an immunoglobulin leader sequence to increase immunogenicity. The HIV antigen encoded by the optimized sequence can comprise a signal peptide such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. The HIV antigen encoded by the optimized sequence can be designed to elicit stronger cellular and / or humoral immune responses than a corresponding native antigen.

[0110] The immunogenic composition can induce an immune response in the subject administered the composition. The induced immune response can be specific for at least one HIV antigen.

[0111] In one embodiment, the immunogenic composition can be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination thereof. The immunogenic composition can include a nucleic acid molecule comprising a sequence encoding the HIV antigen in the form ofa self-assembling nanoparticle. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the HIV antigen by a peptide bond.

[0112] In one embodiment, the immunogen can be used as a priming immunogen. In another embodiment, the immunogen can be used as the second, third, fourth, fifth, sixth immunogen of a series.Other Components of the Composition

[0113] In some embodiments, the immunogenic composition of the invention further includes a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can include such functional molecules as vehicles, adjuvants, carriers or diluents, which are known and readily available to the public. Preferably, the pharmaceutically acceptable excipient is an adjuvant or transfection facilitating agent. In some embodiments, the nucleic acid molecule, or DNA plasmid, is delivered to the cells in conjunction with administration of a polynucleotide function enhancer or a genetic vaccine facilitator agent (or transfection facilitating agent). Polynucleotide function enhancers are described in U.S. Serial Number 5,593,972, 5,962,428 and International Application Serial Number PCT / US94 / 00899 fded January 26, 1994, which are each incorporated herein by reference. Genetic vaccine facilitator agents are described in US. Serial Number 021,579 filed April 1, 1994, which is incorporated herein by reference. The transfection facilitating agent can be administered in conjunction with nucleic acid molecules as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules. Examples of transfection facilitating agents includes surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is apolyanion, polycation, including poly-L-glutamate (LGS), or lipid.

[0114] In some embodiments of the present invention, the immunogenic compositions can further include an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of: alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, TNF0, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful adjuvants include those encoding: MCP-1, MIP-l-alpha, MIP-lp, IL-8, RANTES, L- selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PEC AM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL- 18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, INK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof. In some preferred embodiments, the adjuvant is selected from IL-12, IL-15, CTACK, TECK, or MEC.

[0115] The immunogenic compositions according to the present invention are formulated according to the mode of administration to be used. In cases where DNA plasmid vaccines are injectable compositions, they are sterile, and / or pyrogen free and / or particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other polycations or polyanions is added to the formulation.

[0116] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers,or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0117] The transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly- L-glutamate may be present in the composition at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the composition. The composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a poly anion, polycation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0118] The composition can be formulated according to the mode of administration to be used. An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The composition can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.Methods of Delivery of the Composition

[0119] The present invention also relates to methods of delivering the composition to the subject in need thereof. The method of delivery can include, administering the composition to the subject. The mammal receiving delivery of the composition may be human, primate, non-human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

[0120] The composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The composition may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns”, or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound.Method of Treatment

[0121] Also provided herein is a method of treating, protecting against, and / or preventing disease in a subject in need thereof by inducing an immune response against a viral antigen in the subject. In certain embodiments, the invention provides a method of treating, protecting against, and / or preventing at least one of an HIV virus infection or an HIV associated pathology in a subject.

[0122] The method can include administering an immunogenic composition of the invention to the subject. Administration of the composition to the subject can be done using the method of delivery described above.

[0123] The composition dose can be between 1 pg to 10 mg active component / kg body weight / time, and can be 20 pg to 10 mg component / kg body weight / time. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0124] The vaccine can be administered prophylactically or therapeutically. Inprophylactic administration, the vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the vaccines are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0125] The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[0126] The vaccine can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).The vaccine can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose. The formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer. The formulation can include aqueous or oily solutions of the vaccine.

[0127] The vaccine can be a liquid preparation such as a suspension, syrup or elixir. The vaccine can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.Kit

[0128] Provided herein is a kit, which can be used for treating a subject using the method of vaccination described above. In one embodiment, the kit can comprise the vaccine. In one embodiment, the kit can comprise a nucleic acid molecule encoding a modified HIV envelope immunogen of the invention.

[0129] The kit can also comprise instructions for carrying out the vaccination method described above and / or how to use the kit. Instructions included in the kit can be affixed to packaging material or can be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, links to websites, QR codes, electronic storage media (e.g., magnetic discs, tapes, cartridges), optical media (e g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.EXAMPLES

[0130] The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within the scope of the appended claims.EXAMPLE 1: DEEP MINING OF THE HUMAN ANTIBODY REPERTOIRE IDENTIFIES FREQUENT AND IMMUNOGENETICALLY DIVERSE CDRH3 TOPOLOGIES TARGETABLE BY VACCINATION

[0131] A major goal of HIV vaccine development is the elicitation of broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env). The Env V2 apex is a promising target for HIV vaccine development, but the identification and engagement of V2 apex bnAb precursors is made difficult by their immunogenetically diverse CDRH3s. However, V2 apex bnAb CDRH3s have been shown to form reproducible structural classes. Sequence bioinformatics was coupled with deep learning structure prediction approaches to investigate the frequency and diversity of one of these classes bearing axe-like CDRH3 shapes within the human repertoire. To elicit axe-like bnAb lineages, a soluble trimer that recapitulates the antigenicity of the Env Q23. 17 was engineered, which naturally exhibits affinity for the inferred germline precursors (iGLs) of this structural class of antibodies. This high-affinity interaction was used to solve the first Cryo-EM structure of the CHOI iGL bound to a native-like trimer (Q23.17 HIV Env). To enhance the antigenicity of the Q23.17 immunogen, nanoparticles were constructed decorated with this trimer, shown that they can be delivered by DNA vaccination, and a novel single-round mammalian display method was used to enhance its antigenicity of the Q23-based construct for all known iGLs of this class. The germline-targeted Q23-based Env immunogens are capable of inducing epitope-specific responses in a human D3-3 / JH6 transgenic mouse model. Finally, infection with a germline targeting Q23-based SHIV can immunofocus B cell responses to the V2 apex and induce heterologous neutralizing antibodies with axe-like properties in rhesus macaques. These results demonstrate that novel approaches coupling deep learning antibody structure prediction across the human BCR repertoire with germline-targeting vaccine design can be employed to create engineered Envs capable of eliciting immunogenetically diverse yet structurally conserved classes of antibodies.

[0132] Following the identification of SARS-CoV-2, a highly efficacious vaccine for the virus was developed in less than a year. Yet, despite over 35 years of research, there is still no protective vaccine for HIV. The HIV envelope glycoprotein (Env), which is the only target for neutralizing antibodies, has several features that make it a challenge for vaccine development.These include a large and dynamic glycan shield, conformational masking of epitopes, and extensive sequence diversity (Stephenson et al., 2020, Annual Review of Immunology 38, 673- 703; Wei et al., 2003, Nature 422, 307-312; Kwong et al., 2002, 420, 678-82; Moore et al., 2001, Journal of Virology 75, 5721-5729). As a result of these properties, the antibody response to Env is usually strain-specific (Moore, 2018, Current HIV research 16, 21-28). However, 10-20% of infected individuals develop broadly neutralizing antibodies (bnAbs) targeting conserved epitopes (Gray et al., 2011, Journal of Virology 85, 4828-4840; Doria-Rose et al., 2010, Journal of Virology 84, 1631-1636; Haynes et al., 2012, Nat Biotechnol, 30, 423-433; Kwong et al., 2018, Cell Press, 48, 855-871; Landais et al., 2016, PLOS Pathogens 12, el005369; Hraber et al., 2014, AIDS (London, England) 28, 163-169; Rusert et al., 2016, Nature Medicine, 22, 1260- 1267), and bnAbs have been shown to be protective against infection by diverse heterologous viruses in humans and rhesus macaques (RMs) (Gautam et al., 2016, Nature 533, 105-109; Corey et al., 2021, New England Journal of Medicine 384, 1003-1014; Julg et al., 2017, Science Translational Medicine 9, eaall321; Hessell et al., 2009, Nature Medicine, 15, 951-954).

[0133] Among the various classes of bnAbs, antibodies targeting the V2 apex are some of the most frequently elicited during infection, are highly potent, and have relatively straightforward developmental pathways (Landais et al., 2017, Immunity, 47, 990-1003. el009; Doria-Rose et al., 2014, Nature, 508, 55-62; Roark et al., 2021, Science, 371, eabd2638). These features make the apex a promising target for vaccine development. However, to date, vaccination has not been able to elicit V2 apex bnAbs in outbred animal models, and there is a need for immunogens capable of doing so. V2 apex bnAbs primarily rely on their long CDRH3s for binding to Env, forming distinct microdomains that contact several glycans and the C-strand of the VI V2 region (Roark et al., 2021, Science, 371, eabd2638; Willis et al., 2022, Immunity 55, 2149-2167. e9; Lee et al., 2017, Immunity 46, 690-702; Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; McLellan et al., 2011, Nature 480, 336-343; Pejchal et al., 2010, Proceedings of the National Academy of Sciences 107, 11483-11488; Julien, et al., 2013, Proceedings of the National Academy of Sciences of the United States of America 110, 4351-6; Gorman et al., 2020, Cell Reports 31, 107488; Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384.) These CDRH3s can be roughly classified into three categories based on their structural topology: “needle-like” in which P-hairpin CDRH3s bearing sulfated tyrosines at their tips extend into the trimer apex hole (Roark et al., 2021, Science, 371, eabd2638; Willis etal., 2022, Immunity 55, 2149-2167. e9; Lee et al., 2017, Immunity 46, 690-702), “axe-like” or “hammerhead-like” in which CDRH3s form extended -sheet interactions with the C-strand (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; McLellan et al., 2011, Nature 480, 336-343; Pejchal et al., 2010, Proceedings of the National Academy of Sciences 107, 11483-11488; Julien, et al., 2013, Proceedings of the National Academy of Sciences of the United States of America 110, 4351-6), and “combined” which share features of both (Gorman et al., 2020, Cell Reports 31, 107488; Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). Because of these unusual CDRH3s, V2 apex bnAb precursors are estimated to have low frequencies in the naive human B cell repertoire, making priming a major hurdle to their elicitation (Willis et al., 2022, Immunity 55, 2149-2167. e9). This challenge is compounded by the fact that, unlike bnAb precursors to other epitopes, V2 apex bnAbs utilize diverse V, D, and J genes, making it difficult to identify V2 apex bnAb precursors based on sequence features alone (Willis et al., 2022, Immunity 55, 2149-2167. e9). Yet, despite their diverse gene usage, V2 apex bnAbs form reproducible structural classes (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). Given the diversity of the antibody repertoire (Briney et al., 2019, Nature 566:7744), we hypothesize that a myriad of genetically divergent CDRH3s can converge on structurally homologous solutions to engage the V2 apex. Here, it is proposed that V2 apex bnAb precursors can be identified by the structural topology of their CDRH3s; and that by targeting a structural, rather than an immunogenetic, class of antibodies, that diversity can be captured and maximize the chances of successfully engaging rare bnAb precursors (Figure 1A).

[0134] Axe-like V2 apex bnAb lineages are typified by the human bnAb lineages PG9 and CHOI (Bonsignori et al., 2011, Journal of Virology 85, 9998-10009; Walker et al., 2009, Science 326, 285-289). To understand the potential structural plasticity and approximate frequency of axe-like bnAb lineage precursors, large-scale sequence and structure-based screens are first conducted to identify the frequency of CHOI -like, PG9-like, and rhesus axe-like antibodies in the human B cell repertoire. A stable recombinant Env trimer immunogen, Q23.MD39, was then constructed and characterized based on the clade A Env Q23.17 which has natural affinity for the inferred germline precursors (iGLs) of the PG9 and CHOI lineages (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; Bonsignori et al., 2011, Journal of Virology 85, 9998-10009; Voss et al., 2017, Cell Reports 21, 222-235; Liao etal., 2013, Immunity 38, 176-186). It was previously shown that immunization with DNA- encoded native-like HIV trimers can enhance antibody responses compared to conventional protein bolus vaccination of the same trimer in WT mice (Xu et al., 2022, Nat Commun 13, 695). To this end, Q23.MD39 is first delivered using DNA immunization and show that it can assemble into well-formed trimers in vivo. To further enhance the antigenicity of the construct, self-assembling ferritin nanoparticles are engineered decorated with Q23.MD39. These nanoparticles showed enhanced engagement of axe-like iGLs, and could be delivered by DNA vaccination to assemble in vivo. The high affinity of Q23.MD39 for the CHOI iGL is used to solve an atomic resolution structure of the antibody, finding that it retains the unique axe-like CDRH3 conformation. In addition, the immunogenicity of this construct was further enhanced through a single-round mammalian display method that significantly increased its affinity for several axe-like iGLs. As a proof of principle of this approach, it is shown that mutated Q23 Envs can elicit V2 apex-targeted responses in stringent mouse models and elicit axe-like neutralizing antibodies to the V2 apex by infecting rhesus macaques with a Q23 -based SHIV incorporating mammalian display-identified mutations.The experimental results are now described.Estimation of axe-like B cell frequency in the human repertoire

[0135] Currently, four axe-like V2 apex bnAb lineages have been described. These include the rhesus lineages V033-a and 41328-a (Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384), as well as the prototypical human lineages CHOI (Bonsignori et al., 2011, Journal of Virology 85, 9998-10009) and PG9 (McLellan et al., 2011, Nature 480, 336-343). The V033-a, CHOI, and PG9 bnAb lineages are promising targets for vaccine development given their high breadth, potency, and relatively low levels of somatic hypermutation. Even though the CHOI lineage has the second-shortest CDRH3 length of canonical human V2 apex bnAbs, previous estimates of B cell precursor frequency have suggested that CHOl-like B cells may be one of the rarer classes of V2 apex bnAbs (Willis et al., 2022, Immunity 55, 2149-2167. e9). This was primarily due to CHOl’s rare D-J gene pairing and its skewed D gene placement in its CDRH3. For the CHOI bnAb lineage, the major contribution of the D gene to the CDRH3 of the antibody is a “YYGSG” (SEQ ID NO:360) sequence motif that forms the C-terminal section ofthe CDRH3 axe. It was hypothesized that bnAb precursor searching should include two previously unconsidered features: 1) that alternative CDRH3 lengths, J genes, and D gene placement could represent a larger pool of potential precursors, and 2) that only a subset of these sequences can form the requisite CDRH3 shape present in this class of bnAbs. Recent advancements in B cell receptor repertoire sequencing, structure prediction tools, and computational power make it possible to identify these potential bnAb precursors through a structure informatic approach.

[0136] The Observed Antibody Space (OAS) database, updated in 2021 for increased accessibility, contains over one billion antibody sequences, allowing for a rich, in-depth antibody repertoire analysis at a previously unachievable level (Olsen et al., 2022, Protein Sci 31, 141- 146). Furthermore, it is now possible to model the antibody sequences within the OAS database with AlphaFold2 (AF2), which represents a fundamental breakthrough in the structure prediction field (Jumper et al., 2021, Nature 596, 583-589). Sequence searches were conducted within the OAS database to estimate frequencies of potential precursor bnAbs. The first set of searches were focused on the CHOI bnAb lineage. Precursor searches of OAS were conducted using sequence criteria for CHOI published previously (Willis et al., 2022, Immunity 55, 2149- 2167. e9), resulting in an average frequency estimation of about 0.01 precursors per million B cells (‘Search 1’ in Figure IB). As the JH gene amino acids do not make direct Env contacts, the JH gene restriction was removed, and observed 1 precursor per million B cells, and an increase in the number of donors with sequence matches (‘Search 2’ in Figure IB). This drastic increase relative to a previous study (Willis et al., 2022, Immunity 55, 2149-2167. e9) was expected since the IGHJ2 gene utilized by CHOI is the least frequently used JH gene used in humans (Volpe et al., 2008, Immunome Res 4, 3). The frequency then increased significantly to -100 precursors per million B cells when the CDRH3 length (20-40 residues in length), motif register (± 2 to 4 residues) and V gene restrictions were relaxed (‘Search 3-7’ in Figure IB). Using the most relaxed search criteria, ‘Search 7’, a diverse set of 48,713 non-redundant antibody sequences were observed and could identify axe-like CHOI sequences in 33 out of the 37 donors who have >1 million sequences in OAS (Figure 2A). Taken together, these results suggest that CHOl-like bnAb precursors may be more frequent in humans than previously presumed.

[0137] To test whether the sequences found from the OAS searches are predicted to have an axe-shaped CDRH3 fold they were modeled with AF2. First, it was determined that AF2could predict the known axe-like CDRH3 of CHOI to < 2A RMSD (Figure 1 C). To assess which mutations in CHOI were required for AF2 to predict the axe-like structure, an in silica saturation mutagenesis screen was conducted by predicting each point mutation within CHOl’s CDRH3. Interestingly positions D105, D106, and to a lesser extent positions G113, SI 14, G115, led to high structural deviation when mutated, suggesting these amino acids are playing a key role in this axe-like micro-fold of the CDRH3 of CHOI (Figure ID). While AF2 can struggle to model most CDRH3 structures (Valdes-Tresanco et al., 2023, Molecules 28, 3991; Ruffolo et al., 2023, Nat Commun 14, 2389; Yin et al., 2024, bioRxiv [Preprint] 2023.07.05.547832), these data demonstrate that the axe-like CDRH3 structure of CHOI can be predicted with high accuracy.

[0138] Next, it was sought to discover which of the BCR sequences from search criteria 7 could fold into axe-like structures with AF2. In order to automate detection of axe-like structures, a general axe-like CDRH3 feature pattern was developed that was termed ‘tum-beta- turn’ from axe harboring antibodies with known structures (Figure IE, Figure 3A). The ‘turnbeta-turn’ pattern consists of at least two consecutive ‘turn’ residues defined using canonical turn metrics and types (de Brevern, 2016, Sci Rep 6, 33191; Lovell et al., 2003, Proteins 50, 437- 450), followed by two or more residues with ‘beta’ backbone geometry at least 20A away from the start of the CDRH3 and capped by at least two more consecutive ‘turn’ residues. The specificity of this ‘turn-beta-turn’ pattern was validated by searching 610 diverse antibody structures which target many different proteins from the SAbDab database (Dunbar et al., 2014, Nucleic Acids Res 42(Database issue), DI 140-D1146) and found five known axe-like structures harboring a ‘turn-beta-turn’ pattern (CHOI lineage, PDB IDs 3TCL, 3U46, 3U4B, and CAP256- VRC26 lineage antibodies, PDB IDs: 4ORD1, 4ORG), and only three other antibodies which exhibit an axe-like fold (Figure 3B). Of the search 7 sequences, 5,146 of the sequences were found to have axe-like structures. It was found that, out of the donors with >1 million antibody sequences, 10.8% do not have any axe-like sequence or structure shaped antibodies, 18.9% have axe-like sequence signatures without an axe shape and 78.4% have a predicted CHOl-like axe shaped CDRH3. To further support AF2 predictions, molecular dynamics (MD) simulations were used to assess the stability of the structural models. 10 replicates of 250 ns simulations were conducted (aggregate of 2.5 ps) for four AF2 models and CH04 (PDB ID: 3TCL) as a control. The axe structural definition was applied to the simulations to determine the frequency of axe-like structures within each simulation. The CDRH3 maintained an axe-like structure for amajority of the time in the CH04 simulations (Figure IF). Three antibodies that matched the ‘tum-b eta-turn’ structural motif (seql0941, seql5061, seq20621, shown in blue in Figure IF) also maintained an axe-like structure in a majority of the simulations. The antibody that did not match the ‘turn-beta-turn’ structural motif (seql0821, Figure IF) had a drastically lower axe-like frequency compared to CH04. These simulations give us insight into the dynamics of the CDRH3 loops and show that this approach could be a useful tool for verifying the stability of CDRH3s with predicted micro-folds, such as the axe shape.

[0139] The axe-like PG9 CDRH3 has a sequence signature that differs from CHOI, and it was determined how frequently the PG9 sequence and structure were found in human repertoires. The expanded sequence and structure modeling pipeline was employed on PG9 as used for CHOI (Figure 2B). PG9-like sequences were found in 35 out of 37 highly sequenced donors (>1M sequences) (Figure 2B). From the 38,427 PG9-like sequences, 6965k sequences had axe-like structures and that 81% of highly sequenced donors have axe-like antibodies. These searches show that, similarly to CHOI, the human antibody repertoire likely contains an immunogenetically diverse set of PG9-like antibodies (Figure 2C).

[0140] The recent discovery of rhesus-derived axe-like bnAb lineages (VO33-a and 41238-a) provides additional targets for vaccine design (Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). Interestingly, although human V2 apex bnAbs use diverse V, D, and J genes, a marked feature of rhesus V2 apex bnAbs is their shared usage of the IGHD3-15*O1 gene (Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). These bnAbs invariably incorporate a D gene-templated “EDDYG” (SEQ ID NO:357) motif that is absent from D genes in the human repertoire. While humans do not contain a germline D gene that encodes “EDDYG” (SEQ ID NO:357), an outstanding question is how frequently V-D-J rearranged antibodies in the human repertoire contain this rhesus sequence motif. To explore this, repertoire and structural searches were conducted to understand how often an “EDDYG” (SEQ ID NO:357) motif is incorporated into human CDRH3s.

[0141] Three searches were performed using either a larger rhesus D gene sequence of “EDDYGYYT” (SEQ ID NO:358) , the core rhesus D gene motif of “EDDYG” (SEQ ID NO:357), and the motif found in the V033-a lineage “[EG]DDYG” (SEQ ID NO:357 and SEQ ID NO:359) with two or more N-terminal amino acids and six or more C-terminal residues relative to the motif (Figure 4A, Figure 2D). Two donors were observed to harbored antibodieswith the larger rhesus D gene sequence (Figure 4A, Search 1). CDRH3s found in donors Hu- CD1 and Hu-BD3 (Waltari et al., 2018, Front Immunol 9, 628) are strikingly similar to rhesus lineages V033-a and 41328-a, suggesting a subset of the human population may have rhesus-like V2 apex bnAb precursors (Figure 4B). While the full D-gene motif was rare amongst donors, the core D gene motifs were found in 89% and 97% of donors (Figure 4A, Search 2 and Search 3). For CDRH3 lengths of >23, the core D-gene motifs were found at 6 per million and 8 per million antibody sequences. To further probe the immunogenetics of antibodies with these core D-gene motifs, the human D-genes used in antibodies found in Search 2 were cataloged. IGHD4- 11*01, IGHD4-17*01 and IGHD4 / OR15-4a*01 each contained the ‘DYG’ sequence (Figure 4B) and were all heavily enriched in the core D-gene search relative the frequency of these D genes found across OAS (Figure 4C). Search 3 CDRH3s were folded using AF2 and showed that 267 sequences out of the 24,696 sequences form axe-like structures (three axe-like and one non-axe examples are shown in Figure 4D). Indeed, MD simulations of rhesus-like axe CDRH3s demonstrate that this fold is highly stable (Figure 4E). Therefore, despite the nearly universal usage of an IGHD3-15*O1 D gene-derived “EDDYG” (SEQ ID NO:357) motif in rhesus V2 apex bnAbs, the absence of an orthologous human D gene may not limit the value of the rhesus model for V2 apex bnAb immunogen design and testing.

[0142] While only four axe-like V2 apex bnAbs have thus far been described, it is likely that far more exist with these sequence and structure features. Using the four axe-like bnAbs as a reference, 12,378 total axe-like antibody sequences were found. Of the donors with > 1 million antibody sequences in OAS, 1 donor did not have any axe-like CDRH3s, but 97% of donors had antibodies with axe-like sequence motifs and 86% of donors had antibodies with axe-like structural motifs (Figure 4F). Collectively, these studies reveal that not only are axe-like sequence motifs identified at high frequency in the human antibody repertoire, but also that antibodies with predicted axe-like structures are observed in the overwhelming majority of donors, pointing to this structural class of bnAbs as a promising vaccine target.Design, characterization, and nucleic acid delivery of Q23.17-based native-like trimer and nanoparticle immunogens

[0143] To design an immunogen that can engage axe-like V2 apex bnAb precursors, a stable trimeric Env protein that could easily multimerize on nanoparticles and be delivered bynucleic acid platforms while retaining its structure in vivo was developed. To discover a starting immunogen sequence, Envs were sought that have detectable affinity for inferred germline precursors of V2 apex bnAbs, since it was predicted that these may be more favorable for V2 apex priming. Screens of Envs to identify such strains with “permissive” V2 epitopes that have natural propensities for V2 apex iGL engagement have previously been conducted (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; Bonsignori et al., 2011, Journal of Virology 85, 9998-10009; Voss et al., 2017, Cell Reports 21, 222-235). Therefore, a clade A Env, Q23.17 was selected (Poss et al., 1999, Journal of Virology 73, 5255-5264), as the platform for immunogen development, because of its relatively high binding affinity for the iGLs of the axe-like CHOI and PG9 bnAb lineages (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90; Bonsignori et al., 2011, Journal of Virology 85, 9998-10009; Voss et al., 2017, Cell Reports 21, 222-235) and sensitivity to mature V2 apex bnAbs. It has been suggested that factors which may contribute to the unique sensitivity of this Env to V2 bnAbs and iGLs include its glycan-devoid V2b loop, and the lack of glycan at N130 (Voss et al., 2017, Cell Reports 21, 222-235). Note that this Env naturally contains many of the “Apex-GT5” residues previously reported to sensitize the Env BG505 to precursors of the PCT64 lineage (Willis et al., 2022, Immunity 55, 2149-2167.e9).

[0144] In order to design a stabilized recombinant Q23.17 trimer, several published Env trimer stabilization strategies were tested including MD39 (Steichen et al., 2016, Immunity 45, 483-496), MD64 (Kulp et al., 2017, Nat Commun 8, 1655), v5 (Pena et al., 2017, Cell Rep 20, 1805-1817), 7S (Antanasijevic et al., 2020, PLoS Pathog 16, el008665), RnS-DS (Rawi et al., 2020, Cell Reports 33, 108432-108432; Rutten et al., 2018, Cell Rep 23, 584-595), and Olio6 (Kulp et al., 2017, Nat Commun 8, 1655) mutations. The Q23.MD39 and Q23.7S demonstrated superior expression and antigenicity. The MD39 mutations are a subset of the 7S mutations, and thus it was concluded that the MD39 mutations were primarily responsible for the stable phenotype of the trimer, focusing on Q23.MD39 for further investigation. Q23.MD39 bound strongly by ELISA to the trimer-specific or trimer-preferring mAbs PGDM1400, PGT151, and VRC34 and bound poorly to V2p-, CD4i-, and linear V3-specific non-neutralizing antibodies, indicating an antigenic profile consistent with a stable prefusion trimer (Figure 5A). In contrast, Q23.gpl20.foldon, a construct consisting of three gpl20 domains loosely held together by a foldon trimerization domain, showed high binding to non-nAbs and poor binding to quaternarystructure-dependent bnAbs (Figure 5 A). The size and homogeneity of Q23.MD39 was determined by size exclusion chromatography multiple angle light scattering (SEC-MALS) which showed that Q23.MD39 formed trimers of the expected size (Figure 6B).

[0145] There is evidence that the multimeric display of vaccine antigens significantly enhances their immunogenicity through avidity effects and enhanced trafficking, among other effects (Kato et al., 2020, Immunity 53, 548-563. e8; Tokatlian et al., 2019, Science 363, 649- 654; Xu et al., 2019, Curr Opin Immunol 59, 49-56; Kelly et al., 2019, Expert Rev Vaccines 18, 269-280). It has previously been shown that DNA-launched nanoparticles (DLNPs) are capable of self-assembly in vivo and are highly immunogenic (Xu et al., 2020, Adv Sci (Weinh) 7, 1902802). To this end, Q23.Ferritin, a self-assembling ferritin nanoparticle decorated with the Q23.MD39 trimer was engineered using a short glycine-serine linker (Figure 14). Using NS-EM and antigenic profiling by ELISA, it was shown that this nanoparticle retains the well-formed phenotype of the Q23.MD39 trimer, expresses primarily as a well-formed nanoparticle by SEC, and has significantly enhanced binding to the axe-like iGLs (Figure 5B-C, Figure 6C). The affinity of the design in trimeric, nanoparticle and gpl20 forms for the CHOI, PG9, V033, and 41328 iGLs demonstrated that Q23-based immunogens have affinity for axe-like iGL precursor antibodies (Figure 5C).

[0146] DNA delivery of HIV Env trimers is a promising approach for driving unique antibody responses in WT mice (Xu et al., 2022, Nat Commun 13, 695). Importantly, SEC analysis showed that Q23.MD39 expressed primarily as a trimer (Figure 5A). Unlike purification of recombinant Env immunogens, nucleic acid delivery strategies are unable to purify out poorly folded or unassembled Env proteins. To show that the trimer was capable of homogenous assembly and expression in vivo, WT BALB / c mice were immunized with a DNA vaccine encoding the Q23.MD39 construct. Mice were immunized with 5pg, lOpg or 25pg of DNA encoded Q23.MD39 with or without adjuvanting with plasmid IL-12 at weeks 0, 3 and 6, following which antibody responses were tracked 2 weeks following each immunization (Figure 5D, Figure 7A). The immunized animals developed strong binding antibody responses, demonstrating that the trimer was immunogenic when delivered as a DNA vaccine (Figure 5D, Figure 7A). However, they failed to develop neutralizing antibody responses, likely due to the absence of the N465 glycan hole previously shown to be the target of nAbs in mice (Xu et al., 2022, Nat Commun 13, 695). To determine if the trimer structure and concomitantly theantigenicity of Q23 MD39 produced in vivo was maintained, the Antigen Conformation Tracing In Vivo (ACTIV) assay was performed (Xu et al., 2022, Nat Commun 13, 695) (Figure 5E). Mice were administered with DNA containing Q23.MD39 or BG505.MD39. In vivo assembled trimers from harvested muscle tissue of mice were analyzed by ELISA for binding to various bnAbs, non-nAbs, and the CHOI iGL at several timepoints post-administration (Figure 7B). High binding of muscle homogenate expressing Q23.MD39 to trimer-specific bnAbs such as PGDM1400, and low binding to non-nAbs such as 17B, indicated that Q23.MD39 retains its favorable antigenic profile when expressed in vivo (Figure 5E, Figure 7B). BG505.MD39, a similarly stabilized and closely related Env, showed a similar binding profile to Q23.MD39. However, only Q23.MD39 was able to bind to CHOI iGL in vivo (Figure 5E, Figure 7B). Escalating-dosing immunization regimes delivered over the course of a few weeks have been shown to significantly improve immune responses through an increase in durable germinal center responses (Bhagchandani et al., 2023, bioRxiv [Preprint] 2023.11.20.563479; Cirelli et al., 2019, Cell 177, 1153-1171. el 128). Here, with DNA delivery, escalating amounts of intact prefusion Env trimer in muscle tissue for at least 3 weeks post-immunization were detected (Figure 5E, Figure 7B). This data shows that Q23.MD39 retains its sensitivity for V2 bnAb iGLs in vivo and marks the first direct demonstration of the ability of an Env trimer produced in vivo to engage a bnAb precursor.

[0147] It was next determined whether Q23.Ferritin could also be delivered by DNA vaccines. DNA immunizations can be used to deliver self-assembling nanoparticle immunogens such as eOD-GT8 60mer (Xu et al., 2020, Adv Sci (Weinh) 7, 1902802), it has been shown that Env trimer nanoparticles can be delivered by mRNA (Mu et al., 2022, Cell Rep 38, 110514). The favorable expression profile of Q23.Ferritin combined with the use of a genetic fusion approach again allowed for delivery of the immunogen via DNA immunization (Figure 6C). DNA delivery of the Q23.Ferritin construct with or without plasmid IL-12 in WT mice elicited high-titer binding antibody responses, demonstrating that Q23. Ferritin is immunogenic when delivered by DNA vaccination (Figure 7A).Cryo-EM structure of Q23.MD39 bound to the CHOI iGL reveals key molecular contacts of axe-like precursor antibodies

[0148] To gain structural insights into how Q23.MD39 binds to the axe-like CHOI iGLwith high affinity, atomic resolution cryo-EM structures were determined of Q23.MD39 in complex with 35022 alone (3.13A) and in complex with the CHOI iGL and 35022 (3.22A) (Figure 9A). A prefusion-closed Q23.MD39 Env trimer was observed with high structural similarity to BG505.SOSIP.664, further indicating that Q23.MD39 is a well-formed native-like trimer. Overlay of the CHOI iGL antibody structure with mature antibody structures of lineages members CH03 and CH04 showed identical conformations for all CDR loops except the CDRL1. This may be due to CHOI iGL lacking the somatic hypermutation of the mature antibodies or because the CHOI iGL is the only antibody structure of this lineage solved in complex with a full Env (Figure 8B). The CHOI iGL antibody bound to Q23.MD39 Env trimer with a 1 : 1 stoichiometry (Figure 8A) and engaged the C-strand using an intricate backbone hydrogen bond network similar to that found in CH03 / CH04 structures in complex with a C- strand scaffolded protein (Figure 8C) (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90). The sidechains of the C-strand on protomer A interact with positions within the axe shape: YlOOg with R169 and YlOOh with K168 (Figure 8C, top middle and top right panels). The CDRH1 may interact with glycan at N156A, although the density for the full glycan could not be built. The sidechains of CDRH1 also interact with both the KI 71 and Y173 on the gpl20A C-strand which are conserved residues among different HIV isolates at these positions (Voss et al., 2017, Cell Reports 21, 222-235) (Figure 8C bottom left panel). The sidechains on the CDRH2 and CDRH3 form extensive contacts with the N160A glycan (Figure 8C bottom middle panel). The structure of the CHOI iGL maintains the axe-like CDRH3 of the mature CHOl-lineage bnAbs, indicating that the unique CDRH3 topology is likely a feature present in the unmutated ancestor of the bnAb lineage. This finding corroborates the structure-guided immunoinformatic approach to bnAb precursor identification conducted in this study, as it suggests that CHOI -like germline precursors may also harbor this unique CDRH3 shape.

[0149] Structures of mature CH03 and CH04 were previously determined using trimeric scaffold mimics with CAP256.SU (CH03) and A244 (CH04) V1 / V2 sequences (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90). The key heavy chain epitopeparatope interactions identified in their structures were very similar to the CHOI iGL structure determined using a trimeric Env. However, due to the unnatural trimeric scaffold protein used in CH03 / CH04 structures, the light chain interactions were unclear. One notable feature, that was not revealed in previous structures, was the close proximity between CDRL2 and the loop V2bfrom gp!20B (Figure 8C, bottom right panel). The N139 glycan, proposed as a second N160 glycan mimic (Gorman et al., 2016, Nature Structural and Molecular Biology 23, 81-90), binding to the HC / LC interface in the CH03 / CH04 structures is missing from the CHOI iGL structure. In the CHOI iGL structure, however, it is unclear if the N160B glycan would occupy the same HC / LC interface space. Although only the first sugar residue of the N160B glycan tree was observed from the density map, there is a slight shift in the position of the glycan from the N139 glycan in the crystal structure and the volume of space between the framework interface of HC / LC is not likely large enough to fit N160B (Figure 8D). However, it was observed by glycan molecular modeling that N160B may be capable of making favorable interactions between CDRL1 and the back of CDRH3 (Figure 8D, bottom right panel).

[0150] Through this structural analysis, there are three potential major contributing factors to the high affinity of Q23.17 for the axe-like CHOI iGL: (i) C-strand residues that are optimal for interactions with the CHOI iGL CDRH3, (ii) a lack of disfavored glycans at the apex epitope such as N130 and loop V2b glycans as previously reported (Voss et al., 2017, Cell Reports 21, 222-235), and (iii) the length of loop V2b being within the threshold accommodated by the CHOI iGL, in particular by CDRL2. This observation suggests V2b could be a promising location for incorporating designed loops or mutations, with the closest contact in the structure being CHOI iGL HC YlOOh and gpl20B E185 (4.82 A). With this structure, one can now visualize an axe-like precursor antibody engaging with the C-strand and further rationalize potential mutations that may increase affinity for CHOI -like axe shaped precursors.Mammalian display mutagenesis to enhance axe-like iGL binding

[0151] Despite the affinity of Q23.MD39 for axe-like iGLs, further modification of the Env is likely required to elicit similar antibodies with high efficiency. The iGLs available for axe-like human bnAb lineages contain affinity matured non-templated regions which comprise a significant portion of the epitope-paratope interface, and so true naive B cell precursors are likely to have lower affinities for Env. To this end, a mammalian display mutagenesis approach was used to enhance the affinity of Q23.MD39 for axe-like iGLs. A previous mammalian display protocol45was optimized by performing single round sorting followed by next-generation sequencing. Briefly, Q23.MD39 was membrane anchored using a PDGFR transmembrane domain and a lentiviral scanning NNK library spanning Env residues 110-192 was created withsilent barcoding mutations flanking each NNK codon. The top 5% of cells were sorted against axe-like iGLs and deep sequenced (Figure 9A). To identify affinity-enhancing mutations, the frequency of particular mutants in the sorted population was compared to the frequency of the same mutation in unsorted cells. This method allowed for identifying many more affinity enhancing mutations far faster than traditional mammalian display mutagenesis approaches (Figure 9B).

[0152] As a proof of principle, the Q23 library was sorted following staining with the V033 iGL. Sequencing of sorted cells led to the identification of mutations spanning the length of the library that were either enriched or selected against, providing an array of potential affinity enhancing mutations (Figure 9B). From these enrichments, positions with low numbers of counts among either the selected or unselected cells, enrichments that arose from low numbers of a mutation appearing in the unsorted library, mutations that occurred at positions where the WT amino acid was also enriched, and mutations that altered or introduced cysteine or proline residues were filtered.

[0153] A subset of 24 especially enriched mutations that passed these criteria were synthesized as soluble Q23.MD39 trimer and then expressed, purified and tested for enhanced binding against the V033 iGL. The majority of the mutations conferred increased binding to the antibody by ELISA ranging from low (<1.5 fold) to high (>3 fold) increase in binding (Figure 9C). It was next asked whether these mutations have an additive enhancement of binding affinity when combined. High binding mutations, Ml 6 IT, L165F, and KI 68V were selected and progressively incorporated into Q23.MD39. ELISA binding to the V033 iGL showed increases in affinity as each mutation was incorporated (Figure 9D). Subsequent addition of a KI 17A, identified through sorts against other axe-like antibodies, further increased the affinity of the V033 iGL, ultimately resulting in a construct named Q23.V033GT. Binding against V033 iGL was assessed by SPR and found that the unmodified Q23.MD39 did not have measurable affinity (up to IpM), however, Q23.V033GT bound at a KD of lOOnM (Figure 10).Construction of and infection with a germline targeting SHIV

[0154] It was next determined whether the Q23.V033GT iGL -targeted immunogen would be able to elicit structurally homologous antibodies in rhesus macaques. Germline targeting mutations were introduced into a replicating SHIV (Li et al., 2021, 1 Virol95, e00071-21; Li et al., 2016, Proc Natl Acad Sci U S A 113, E3413-22), and the germline targeting capacity was tested through infection rather than immunization. This was done because: (i) the high and sustained antigenic loads during viral infection maximize the probability of engaging bnAb precursor B cells, similar to extended dosing or using nucleic acid delivery; (ii) SHIVs coevolve with the bnAb lineage, selecting for affinity maturation and the acquisition of neutralization breadth, thereby allowing us to verify that primed B cells are genuine bnAb precursors; (iii) the pattern of Env escape can aid in the design of boosting immunogens, since escape variants select for affinity maturation and neutralization breadth; and (iv) sequencing of Env escape serves as a sensitive indicator of nAb-targeted epitopes, allowing identification of both on- and off-target nAb lineages (Roark et al., 2021, Science, 371, eabd2638; Bibollet-Ruche et al., 2023, mBio 14, e0337022).

[0155] In order to construct this germline targeting SHIV, the mammalian display- identified mutations were incorporated into the WT SHIV-Q23 (Li et al., 2021, J Virol 95, e00071-21). It was determined that the V033 -targeting mutations in Q23.V033GT could be incorporated into the virus without impacting viral infectivity (Figure 11 A). A SHIV bearing all 4 of these mutations, SHIV-Q23.V033GT maintained infectivity and p27 antigen titers similar to WT (Figure HA). Strikingly, neutralization assays showed an over 50-fold increase in neutralization potency by the V033 iGL against the resulting SHIV-Q23.V033GT, verifying that the germline targeting mutations retained their effect in the context of an infectious virion (Figure 12A). Antigenic profiling of the germline targeting SHIV demonstrated that the Q23.V033GT Env retained a closed prefusion state, showing no sensitivity to a panel of CD4i and V2p antibodies consisting of CH58, CAP228-3D, 447-52D, 17b, A32, 697-D, and 1393. The SHIV does show a modest increase in neutralization sensitivity to the antibody 3074, suggesting a more exposed V3 loop (Figure 12A).

[0156] Three rhesus macaques were infected with SHIV-Q23.V033GT, as previously described (Roark et al., 2021, Science, 371, eabd2638; Li et al., 2021, J Virol 95, e00071-21). All rhesus macaques were productively infected, with peak viral loads between 107- 108vRNA copies / ml of plasma 2 weeks post infection (Figure 1 IB). Viral loads for one rhesus macaque, CL60, were especially high during the first 12 weeks of infection (>5xl06vRNA copies / ml). This animal failed to develop any detectable antibody response to SHIV infection (negative HIV / SHIV ELISA, western immunoblot, autologous nAb titer) andsuccumbed to rapid clinical progression of AIDS (Brown et al., 2007, J Virol 81 , 5594-5606) (Figure 1 IB). The other two macaques exhibited stable setpoint viral loads between 5xlO4-5xlO5vRNA copies / ml (Figure 1 IB). Each mounted autologous Q23.V033GT and tier 1A nAb responses by 12 weeks post infection (Figure 12B). Rhesus macaque CK52 failed to neutralize WT Q23.17 at week 12 post-infection, suggesting that the entire nAb response was immunofocused to the V2 apex and dependent on the germline targeting mutations introduced (Figure 12B). In both CK11 and CK52, we measured neutralization of V2 apex site-directed Q23.17 mutants (N160K and N187S) and observed a striking enhancement or reduction in neutralization, thus confirming the presence of C-strand targeted nAb responses (Figure 12B). Single-genome sequencing in CK52 and CK11 showed strong selection at the V2 apex at week 12 post-infection, with the addition of glycans to the V2b loop and selection for neutralization escape at C-strand residues 170-172 (Figure 12C, Figure 11D-E).

[0157] In order to understand how efficiently these C-strand targeted nAbs were being primed and affinity matured by SHIV.Q23.V033GT compared to SHIVs bearing wild-type Envs, the kinetics of Env escape in macaques CK52 and CK11 were compared with 6 macaques infected with WT SHIV-BG505.N332 and 4 macaques infected with WT SHIV-Q23.17 (Figure 12C-D). Kinetics of epitope escape was used as a measure of C-strand targeted nAb elicitation, since virus escape has been shown to precede or coincide with the first detection of epitope specific nAbs (Bar et al., 2012, PLoS Pathog 8, el002721; Wang et al., 2024, Cell 187, 7214- 7231 e23). Week 12 was selected for the comparison because it is the first timepoint where autologous nAbs were detectable during infection, and therefore indicative of an Env’s ability to immunofocus early responses to particular epitopes. At this timepoint, none of the SHIV- BG505.N332 or SHIV-Q23.17 infected RMs exhibited escape from a V2 apex targeted nAbs in the form of C-strand mutations or V2b glycan additions (Figure 12C). This demonstrates that SHIV-Q23.V033GT was able to prime responses to the V2 apex more efficiently than these two primary HIV Envs (Figure 12C, V2 apex escape mutations found in 4 of 232 BG505 sequences, 1 of 113 Q23.17 WT sequences and 10 of 66 Q23.V033GT sequences). Further, escape mutations were quantified by V2 apex Hamming distance measurements relative to starting SHIV sequence and observed that SHIV Q23.V033GT elicited the most epitope specific mutations compared to SHIV-BG505.N332 or SHIV-Q23.17 (Figure 12D). These findings demonstrate that a germline targeted SHIV with enhanced affinity for V2 apex precursors canconsistently elicit V2 apex C-strand targeted nAbs early in infection.

[0158] To determine if the macaques with V2 apex responses also generated neutralization breath, neutralization activity was measured against a panel of nine heterologous tier 2 viruses. Heterologous neutralization was detectable in both RMs CK11 and CK52 at 28 weeks post-infection. Both plasma samples neutralized BG505 at titers of 1 :20 to 1 :30. CK52 plasma also neutralized C1080 at a titer of 1 :200 and CH505 at a titer of 1 :20 (Figure 12E). For both monkeys, heterologous plasma neutralization was mapped to the V2 apex C-strand using site-directed mutants (Figure 1 IF). Finally, a powerful imaging technique, Epitope Mapping by Polyclonal Electron Microscopy (EMPEM), was employed using both negative stain (nsEMPEM) (Bianchi et al., 2018, Immunity 49, 288-300. e8) and cryo-EM (cryoEMPEM) (Antanasijevic et al., 2021, Nat Commun 12, 4817) to confirm the presence of V2 apex directed antibodies in SHIV immune sera. The nsEMPEM data revealed strongly epitope focused responses, as we observed only V2 apex and gp41 binding antibodies at wk24 in RM CK11 and only V2 apex antibodies in RM CK52 (Figure 12F, Figure 26A, B). Further, multiple V2 apex targeting antibodies with an off-center angle of approach characteristic of axe-like bnAbs were observed (Figure 12F). By fitting high resolution Env-axe antibody complexes into the CK52 nsEMPEM density, significant overlap was observed with that of the V033, 41328 and CHOI iGL Fabs, indicating the presence of a structurally homologous antibody (Figure 12G). Next, cryo-EMPEM was performed on the CK52 sample at week 24 and obtained a 4.2 resolution structure. Consistent with nsEMPEM results, the cryo-EMPEM confirmed V2 apex engagement targeting the C strand region similar to the approach of axe-like bnAbs (Figure 12H). Further refinement revealed beta strand density likely contributed by the antibody CDRH3 interacting with the C-Strand as expected for axe-like engagement of Env V2 apex. Together, the escape mutations, neutralization sensitivity and structural data demonstrate that SHIV-Q23.V033GT reelicited axe-like antibodies to the Env V2 apex early in infection.Construction of and immunization with a pan-axe targeting immunogen

[0159] It was next sought to simultaneously enhance the affinity of Q23.MD39 for all known axe-like iGL antibodies. An immunogen with increased affinity for multiple immunogenetically distinct iGLs of this class would be generally more efficient in binding naive germline B cell precursors harboring this CDRH3 topology. To do this, the large number ofenrichments identified by the mammalian display approach was used. By generating matrices of enrichments following sorting of the NNK library against several iGLs of interest and looking for enrichments in common, a set of mutations were identified that simultaneously increased affinity for the V033, 41328, PG9 and CHOI iGLs (Figure 13A-B, Figure 14). The mutations in this trimer, Q23.RH-GT (Rhesus-Human Germline Targeting), were all distal to the C-strand, the primary point of contact at the epitope-paratope interface, and instead may enhance affinity through second shell interactions or indirect restructuring of the Env apex, making it more amenable to engagement by axe-like antibodies.

[0160] To test the ability of this immunogen to elicit axe-like bnAb precursor lineages, hD3-3 / JH6 mice (Schiffner et al., 2024, Nat Immunol 25, 1073-1082) were immunized. This model has the human IGHD3-3 and IGHJ6 genes knocked in to the mouse DQ52 and JH1-4 loci respectively, allowing the B cells to incorporate these genes during VDJ recombination and produce diverse CDRH3s of lengths longer than WT mice and comparable to human V2 apex bnAbs that incorporate these same genes (Schiffner et al., 2024, Nat Immunol 25, 1073-1082). The hD3-3 / JH6 mice(n=3) were immunized with 25pg of DNA encoding an RH-GT trimer along with IL-12 adjuvant, and boosted 3 weeks post prime (Figure 13C). All mice showed strong binding to the Q23. RH-GT trimer but diminished binding to a V2 apex C-strand epitope knockout trimer (Q23.MD39 R169E / K71E), indicating the presence of robust C-strand directed responses (Figure 13C). Control mice immunized with Q23. ferritin immunogen containing a WT V2 apex epitope. These mice failed to induce the same level of epitope-specific response. These data indicate that the Q23.RH-GT immunogen was successfully able to induce V2 apex responses in a stringent rearranging mouse model.Discussion

[0161] Germline targeting approaches have successfully primed HIV bnAb precursors for lineages where the bnAb paratope consists of mostly germline-templated amino acids (Leggat et al., 2022, Science 378, eadd6502). However, bnAbs with CDRH3 -dominated modes of binding have proven more challenging, with V2 apex bnAbs being a particularly difficult target given their long CDRH3s containing extensive non-templated regions. Groups have previously described exciting approaches to eliciting antibodies with predominantly CDRH3 -mediated binding through the design of immunogens that bind B cells with strictly defined criteria,consisting of invariant V, D, and J gene templated regions but diverse non-templated junctions (Steichen et al., 2019, Science 366, eaax4380). A major limitation of these approaches is that they are predicated on the selection of a suitable template antibody, and are therefore limited by the antibody lineages available for targeting. Here this immunogen design approach is expanded by instead targeting a structural class of V2 apex bnAbs, leveraging immunogenetically diverse but structurally homologous antibodies to optimize a trimer for axe-like V2 apex bnAb elicitation.

[0162] These findings suggest that the frequency of antibodies with axe-like CDRH3 topologies is likely far higher than previous estimates based solely on the immunogenetic features of B cells (Willis et al., 2022, Immunity 55, 2149-2167. e9). This is built on previous work by leveraging recent advancements in deep learning structure prediction to incorporate predicted B cell CDRH3 topology into immunoinformatic searches. Three key observations support the structurally focused approach to precursor targeting. First, the structure of the CHOI iGL retains its distinct axe-like CDRH3 topology, as was seen with PG9 iGL (Willis et al., 2022, Immunity 55, 2149-2167. e9). This suggests axe-like precursors acquire their distinct CDRH3 conformation during VDJ recombination, as opposed to through affinity maturation, and that additional precursors may be identified by the presence of this distinct CDRH3 conformation. Second, it is demonstrated that AlphaFold2 is capable of folding axe-like CDRH3 structures and that a structurally conserved ‘turn-loop-turn’ definition can be used to identify new axe-like CDRH3s in an automated fashion. The third observation is the recent isolation of rhesus V2 apex bnAbs that recapitulate the axe shape found in C-strand targeting human V2 apex bnAbs despite using rhesus-specific D genes (Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). This structural homology underscores the ability of immunogenetically diverse CDRH3s to adopt similar solutions to C-strand recognition. Furthermore, the identification of B cells bearing the key rhesus “EDDYG” (SEQ ID NO:357) motif in the human repertoire supports the use of rhesus V2 apex bnAb precursors for human vaccinology. With the advent of more accurate protein structure prediction tools such as AF3 (Abramson et al., 2024, Nature 630, 8016) and Ig- Fold (Ruffolo et al., 2023, Nat Commun 14, 2389), the new approach will provide an increasingly more powerful method for further precursor analyses.

[0163] These data show that germline-targeting mutations can be engineered into Env Q23.17 in order to elicit V2 apex-specific responses in mouse models with long CDRH3s andinduce antibodies that target the V2 apex with axe-like engagement and neutralize heterologous tier-2 viruses in rhesus macaques. These data suggest that the HuD3-3 / J6 mouse model may be of utility to drive V2 apex-specific responses. The SHIV infection model has been employed to study Env-antibody coevolution resulting in identification of bnAbs that recapitulate sequence and structural features of human bnAbs (Roark et al., 2021, Science, 371, eabd2638; Roark et al., 2024, bioRxiv [preprint] 2024.06.11.598384). This platform was exploited for evaluation of germline-targeting Env. In both evaluable remaining monkeys, the GT Env was able to rapidly elicit escape mutations at the V2 apex and drive limited heterologous neutralization. These studies should be followed up with rationally designed booster immunogens in heterologous vaccination experiments.

[0164] In summary, it is possible to design immunogens that target the broad axe-like structural class of CDRH3s. The Q23.RH-GT is such an immunogen that has improved affinity for all four axe-like inferred germline antibodies with the ability to activate similar antibodies in animal models. It is key to determine the frequency of B cells harboring a defined structural class of CDRH3s, as it was shown here that axe-like precursor antibodies are present in most people. As the axe microdomain is not the only structural class of CDRH3s, this structure-guided immunoinformatic approach will open the door for many new studies on additional important epitopes on HIV-1 Env, as well as other glycoproteins with rich sets of antibody structures such as SARS-CoV-2, Influenza and RSV.METHODSMD simulations

[0165] Molecular dynamics simulations were seeded from AlphaFold2-generated structures using the GROMACS software and the CHARMM36m force field, a general force field that has been shown to work for antibodies. The structures consisted of a variable heavy chain and the mature variable light chain for the respective bnAb (CHOI, PG9, V033). The TIP3P water model was used, with 0.1 pM NaCl. Energy minimization was performed using steepest descent minimization, followed by NVT equilibration for 200 ps, NPT equilibration for 1 ns, and three 50 ns production runs per sequence. Analysis was conducted using the MDTraj and MSL software.In-silico saturated mutagenesis

[0166] In-silico saturated mutagenesis was conducted using AlphaFold2 and mutating each residue on the CHOI CDRH3 to every possible amino acid. Each prediction had 5 models and 10 seeds per model, resulting in 50 structures per mutation. The RMSD values were computed between the CDRH3 regions of the CHOI AlphaFold2 structure and the AlphaFold2 predictions of the mutations. Code will be available on Github for all steps.DNA design and plasmid synthesis

[0167] The amino acid sequence for Q23.17 was obtained from a previously published sequence (genbank accession number AF004885.1). Stabilized trimer constructs were developed by adding published stabilization mutations into the Q23.17, and all constructs included the T533A “repair” mutation. Constructs were then codon optimized, and an optimized IgE leader sequence was added to the N terminus of the protein to provide efficient processing and secretion. All plasmids were synthesized and cloned (GenScript) into a modified pVaxl backbone (Inovio Pharmaceuticals).Antibody expression and purification

[0168] Expi293F cells (ThermoFisher) were maintained in Expi293 expression medium (ThermoFisher). All cell lines were mycoplasma negative and tested on a regular basis. All proteins were produced by Expifectamine transfection of Expi293F cells following the manufacturer’s protocol. Transfection enhancers were added 18 h after transfection and supernatants were harvested 6 days later. Antibodies were purified using Protein A agarose according to the manufacturer’s protocol to purify the IgG. Purity was confirmed with Coomassie staining of SDS-page gels and concentration was determined using a nanodrop.Pseudovirus production and purification

[0169] Pseudotyped viruses were produced using HEK 293 T cells transfected with 4 pg of a plasmid expressing the Env of interest and 8 pg of a plasmid expressing the HIV-1 backbone A Env (pSG3AEnv - NIH AIDS Reagents) using GeneJammer (Aglient). Forty-eight hours after transfection, cell supernatant was harvested, filtered through a 45 pm filter, aliquoted, and stored at -80 °C.Trimer production and purification

[0170] Env-based trimers were expressed in Expi293F cells maintained in Expi293 expression medium (ThermoFisher). Trimers were produced by Expifectamine transfection (Gibco, A14524) of Expi293F cells following the manufacturer’s protocol. The trimer-containing supernatants were obtained by centrifuging (4000 x g, 25 mins) and filtering (0.2 um Nalgene Rapid-Flow Filter) the 293F cultures, following which trimers were purified from supernatants by lectin purification using lectin beads (Vector Laboratories) and lectin elution buffer (IM Methyl alpha-D-mannopyranoside). The trimers were then purified over a size-exclusion chromatography column (GE S200 Increase) in PBS. The molecular weight and homogeneity of the trimers were confirmed by protein conjugated analysis from ASTRA with data collected from a size-exclusion chromatography-multi-angle light scattering (SEC-MALS) experiment run in PBS using a GE S6 Increase column followed by DAWN HELEOS II and Optilab T-rEX detectors. The trimers were aliquoted at 1 mg / ml and flash frozen in thin-walled PCR tubes prior to use.Antigen Conformation Tracing in vivo by ELISA (ACTIVE)

[0171] For Antigen Conformation Tracing in vivo by ELISA, BALB / c mice were administered with 100 pg DNA plasmid co-formulated with 12 U hyaluronidase in the tibialis anterior (TA) muscles of the mice as described previously (Xu et al., 2022, Nat Commun 13, 695). At predetermined time points TA muscles were harvested and homogenized in T-PER extraction buffer (Thermo Fisher Scientific) containing protease inhibitor (Roche). Muscle homogenates were subsequently concentrated using a 3kDa Amicon Ultra 0.5 mL centrifugation kit (Millipore Sigma). Concentrations of total proteins were estimated using BCA assay kit (Thermo Fisher Scientific). In order to determine correct folding and binding of in vivo expressed antigens, 96 well ELISA plates (Coming, 3690) were coated with 4 ug / ml of recombinant PGT128 Fab fragments in PBS and incubated overnight at 4° C. After washing, plates were blocked with 5% skimmed milk in PBS containing 1% newborn calf serum (NBS) and 0.2% Tween for 1 hour at room temperature (RT). Total protein concentrations were normalized and added to the plate followed by serial dilutions. Recombinant BG505.MD39, Q23.MD39, Q23. Ferritin and gpl20-foldon were added as positive control standards. Plates wereincubated at RT for 2 h, then followed by washing, antibodies of interest were added at 10 pg / ml except for PGT145 which was added at 50 pg / ml, for 1 hour at 37 °C. Plates were further washed and incubated with Goat Anti-Human IgG Fc Fragment conjugated with HRP (Bethyl Laboratories Inc) for 1 hour at RT. Plates were developed for 5 min with 1-step ultra TMB (ThermoFisher) and stopped with 1 N H2SO4. Absorbance at an optical density (OD) of 450 nm and 570 nm was measured using Synergy2 plate reader (BioTek Instrument). The background 570 nm OD was subtracted from the 450 nm reading. The data was analyzed and fitted using Graph Pad Prism 10.2.Antibody Digestion for Complexation

[0172] Monoclonal and polyclonal antibodies were digested into antigen-binding fragments (Fabs) by adding antibodies into 6-12 mL of digestion buffer (100 mM sodium acetate 10 mM L-cysteine 0.3 mM EDTA pH 5.6 or 7) followed with the addition of papain (2%, w / w), pre-incubated for 15 mins in digestion buffer. Digestion reactions were quenched using 3 mM iodoacetamide and fabs were purified using protein A resin. Protein A resin was added into digestion mixture and incubated for 15 mins on ice. Digest antibody / Protein A resin mixture was filtered through a gravity column. Flow through was collected and buffer exchanged into IX PBS using Amicon-Ultra concentrator with molecular weight cutoff of 10 kDa. Protein A was resin washed with at least 10 CV of IX PBS and eluted using protein A elution buffer. Flow through, wash, and elution fractions were analyzed by SDS-PAGE. CHOliGL Fabs were unable to be purified using Protein A resin due to unwanted binding of Fab to Protein A. Instead, digestion mixture mixed with 3mM iodoacetamide was buffer exchanged into IX PBS. CHOI iGL Fabs / Fc / IgG mixture was separated using Superdex 200 increase 10 / 300 size exclusion column using IX PBS as the running buffer to purify the CHOliGL Fabs / Fc mixture. Fractions containing CHOliGL Fabs / Fc were concentrated using Amicon-Ultra concentrator with a molecular weight cutoff lOkDa. All Fabs were stored at 4°C.Negative stain electron microscopy sample preparation and data collection

[0173] SEC purified MD39 trimers were further dialyzed into Tris-buffered saline (TBS). A total of 4 pL of purified proteins (0.005 mg / mL) was adsorbed onto glow discharged carbon- coated Cu400 EM grids. The grids were then stained with 4 pL of 2% uranyl formate, blotted,and stained again with 4 pL of the stain followed by a final blot. Image collection was performed on a FEI Tecnai T12 microscope equipped with Oneview Gatan camera at 62,750x camera magnification resulting in pixel size of 2.356 A.

[0174] For nsEMPEM, Q23.GT4 (50 pg) was mixed with 500 pg of polyclonal fabs and incubated at 4 degrees overnight. The resulting complex mixture was purified using Superose 6 increase 10 / 300 size exclusion column and complex fractions were collected. The collected fractions were diluted immediately and applied to glow discharged carbon coated 300 mesh Cu grids (Electron Microscopy Sciences (EMS) CFTH3OO-Cu-5O) to incubate for 2 mins before blotting. Complexes were stained using 2% (w / v) uranyl formate. Micrographs were collected on a FEI Tecnai T12 microscope at 62,750x camera magnification resulting in pixel size of 2.356 A / pixel.Negative stain electron microscopy data processing

[0175] Micrographs were imported into RELION5.0 and performed CTF estimation using CtfFind4. Particles were picked using Laplacian of Gaussian (LoG) picker and extracted for 2D classifications. For negative stain EMPEM, after the final round of 2D classification, there were 209,484 particles in the CK52 dataset and 59,730 particles in the CK11 dataset. These particles were then 3D classified with 50 classes for Ck52 and 60 classes for CK11 (T=4). Good 3d classes were individually 3D refined using a 15 A ligand-free Env density as a reference.Preparation of Q23.MD39 in complex with CHOI iGL and 35022

[0176] Q23.MD39 / CH01iGL / 35O22 complex was prepared by mixing Fabs with a1 :40:40 molar ratio (Q23.MD39:CH01iGL:35O22) in IX PBS. Q23.MD39 complex was purified by Size-Exclusion Chromatography using the ENrich™ 650 column (BioRad) with IX PBS as the running buffer and fractions containing the complex were collected and concentrated to 0.1 mg / mL. Complex was used for negative stain EM analysis immediately and aliquoted for storage in -80°C until Cryo-EM sample preparation.Cryo Electron Microscopy Sample Preparation, Data Collection, and Data Processing

[0177] Q23.MD39 / CH01-iGL / 35O22 sample was diluted to 0.07 mg / mL and deposited on graphene-oxide (GO) coated Au-Flat grids (Protochips). Graphene oxide coating of Au-Flatgrids were done in-house following a protocol from previous literature (Patel et al., 2021, bioRxiv [Preprint] 2021.03.08.434344). 4 pL of sample was added to the GO-coated grid at 4°C under 100% humidity in a Mark IV Vitrobot (FEI), blotted with Whatman #1 fdter paper, and then plunged immediately into liquid ethane. Dose-fractionated data for Q23.MD39 / CH01- iGL / 35022 was collected in Counting mode at a magnification of 81,000x resulting in pixel size of 1.054 A / pixel using aberration-free image shift (AFIS) protocol through the EPU software (ThermoFisher).

[0178] Data processing was performed employing a standard cryo-EM data processing workflow comprising motion correction, CTF estimation, reference-free LoG picking, 2D classification, manual inspection / selection of 2D class averages, and associated molecular projection image data and asymmetric 3D refinement (RELION v3.1). (Zivanov et al., 2018, Elife 7:e42166) 3D classification was employed to remove junk particles as well as separating particles with two and three 35022-bound particles. Particles from two and three 35022-bound Q23 / CH01 iGL classes were individually 3D Refined and Bayesian Polished in RELION. Polished particles were transferred into CryoSPARC and 3D classified to further improve the CHOliGL density. The resulting 3D classes were individually refined, and the best class was manually inspected and used for another round of Non-Uniform Refinement to generate the final density maps.Cryo EMPEM Sample Preparation and Data Collection

[0179] Q23.GT4 (100 pg) were added into 1 mg of CK52 week24 polyclonal Fabs and incubated at 4 degrees overnight. CK52wk24 / Q23.GT4 complex was purified using S6i 10 / 300 size exclusion chromatography in IX PBS and fractions were collected, pooled, and concentrated using Amicon-Ultra concentrator with a molecular weight cutoff of 10 kDa. Complexes were diluted to 0.075 mg / mL and deposited on GO-coated UltrAuFoil Rl.2 / 1.3 grids (Quantifoil). Sample grid was loaded onto a Thermo Scientific Glacios (Thermo Fisher) equipped with a Falcon 4 detector. Dose-fractionated data was collected at 150,000x magnification resulting in a pixel size of 0.95 A / pixel using AFIS protocol through EPU software (Thermofisher). Data was collected with a total dose of 50 e7 A2divided over 45 frames.Cryo EMPEM Data Processing

[0180] Micrographs were first imported into RELION (v5.0) (PMID: 22100448) for motion correction and then subsequently, transferred into cryoSPARC for Patch CTF Estimation. Blob picker was used for particle picking and extracted (binned by 4) for iterative rounds of 2D classification and subsequently, ab initio reconstruction. The initial reconstructed density map was then used as a template for template picking. Particles were extracted (binned by 4) and 2D classified, and trimer particles were further filtered using heterogenous refinement using the initial density map as well as ab initio reconstructions of junk particles. Particles were reextracted (binned by 2) and another round of Non-Uniform refinement was performed. Finally, particles were re-extracted with the original pixel size and were Non-Uniform refined again. Selected particles were subjected to 3D classification using an 80 A sphere mask covering the apex of the Env trimer (K=##). Particles in the resulting class with the fab density were subjected to a final round of non-uniform refinement.Model Building

[0181] A homology model of Q23.MD39 was generated from SWISS-MODEL (Schwede et al., 2003, Nucleic Acids Res 31, :3381-3385) using a refined model of a high- resolution prefusion-closed Env trimer. A homology model of CHOl-iGL Fab was generated from SWISS-MODEL using CH03 Fab (PDB: 5ESV) (Gorman et al., 2016, Nat Struct Mol Biol 23, 81-90). Crystal structure of 35o22 Fab (PDB: 4TOY) was used. Homology models were docked into the Q23.MD39 / CH01iGL(A) / 35o22(2) density map (UCSF ChimeraX) (Meng et al., 2023, Protein Sci 32, e4792) and real-space refined using Coot (Emsley et al., 2004, Acta Crystallogr D Biol Crystallogr 60, 2126-2132). The resulting protein build was refined using Rosetta with in-house developed protocols. N-linked glycans were added manually in Coot and the protein model was refined for a total of six times (Rosetta). The Q23.MD39 / CH01iGL(A) / 35o22(2) atomic model was docked into the Q23.MD39CH01iGL(A) / 35o22(3) density map with an additional 35o22 Fab, and the model was manually adjusted in Coot as well as globally refined in Rosetta. Q23 / 35o22(2) atomic model was refined using the same protocol. Model geometry was validated using MolProbity (Chen et al., 2010, Acta Cryst. D66, 12-21) glycan geometry using Privateer31 (Agirre et al., Nat Struct Mol Biol 22, 833-834), and model-to-map fit using EMRinger (Barad et al., 2015, Nat Methods 12, 943-946).ELISAsSerological trimer-binding ELISA

[0182] Binding titers to trimer were determined by coating plates with 2 pg / ml of recombinant PGT128 antibody overnight in PBS. After washing, plates were blocked with 5% skim milk in PBS with 1% newborn calf serum (NBS) and 0.2% Tween for 1 hour at RT. Recombinant trimer was added at 4 pg / ml for 2 hours at RT. Serum was serially diluted, added to plates, and incubated at 37 °C for 1 hour. Antigen and species-specific IgG was then detected across absorbed secondary anti-mouse HRP antibody (Bethyl Laboratories Inc). Plates were developed for 5 min with 1-step ultra TMB (ThermoFisher) and stopped with 1 N H2SO4. Absorbance at an optical density (OD) of 450 nm and 570 nm was measured on a Synergy2 plate reader (BioTek Instrument). The background 570 nm OD was subtracted from the 450 nm reading.Recombinant antibody binding ELISA

[0183] Antibody affinity to Env trimers was determined by coating plates with 4 pg / ml PGT121 or PGT128 fab in 1 *PBS for 3 hours at RT. After washing with 1 *PBS containing 0.05% Tween, plates were blocked overnight with 1 *PBS containing 0.1% Tween and 5% skim milk. Plates were washed and trimer was incubated at 10 pg / ml for 1 hour at RT. Plates were washed and recombinant antibody was serially diluted, added to plates, and incubated for 1 hour at RT. After washing the plates, goat anti -human IgG Fc (Bethyl Laboratories Inc) at a dilution of 1 : 10,000 was incubated for 1 hour at RT. Plates were washed and developed for 10 min with 1-step ultra TMB (ThermoFisher) and stopped with 1 N H2SO4. Absorbance at an optical density (OD) of 450 nm and 570 nm was measured on a Synergy2 plate reader (BioTek Instrument). The background 570 nm OD was subtracted from the 450 nm reading.DNA encoded immunogen immunization in WT (BALB / c) mice

[0184] Female BALB / c mice of 6-8 weeks old were immunized with 5 pg, 10 pg and 25 pg of plasmid DNA encoding Q23.MD39 trimer or 5 pg and 10 pg of Q23. Ferritin nanoparticle. The animals received these immunizations either with or without plasmid DNA encoding IL- 12 as intramuscular (IM) injections into the tibia anterior (TA) muscles, followed by in vivoelectroporation (EP) using the CELLECTRA ® -3P device (Inovio Pharmaceuticals). Mice were immunized at 0, 3 and 6 weeks and sera were collected 2 weeks post each immunization through the submandibular vein for assessment of humoral immune responses.Neutralization Assays

[0185] Pseudotyped viruses and SHIVs were titered on TZM-bl cells to determine lU / ml. All sera samples were heat-inactivated for 60 min at 56°C. 96 well plates were seeded with 104TZM-bl cells / well cultured in DMEM + 10% FBS one day prior to the assay. Serum was serially diluted in media supplemented with 10% normal human serum and incubated with pseudotyped virus and dextran (ThermoFisher) for 1 hour at 37°C, following which the virus / serum mixture was added to adherent TZM-bl cells. Forty-eight hours after incubation, media was removed and cells were lysed using PBS + 0.01% Triton-X (Promega). Luciferase luminescence was then measured using the Synergy2 plate reader (BioTek Instruments). Serum titer was determined for 50% virus neutralization (ID50).Mammalian display library design

[0186] For cell surface display, Q23.MD39 was genetically fused to a PDGFR transmembrane domain via a G / S linker as previously described (Steichen et al., 2016, Immunity 45, 483-496). This construct was synthesized in a pENTR backbone by Twist Biosciences. A scanning NNK library covering HXB2 residues 110-192 of the Q23.MD39 construct was produced using a BioXP 3250 instrument. NNK fragments were then pooled and assembled into the Q23. PDGFR. pENTR construct via Gibson assembly (NEB, E261 IS). The Gibson reaction was then transformed into Stbl2 cells, and a small aliquot of the transformation culture was plated to ensure a transformation efficiency of >10X the library diversity. Library assembly and diversity was confirmed by sequencing on an Illumina MiSeq.Mammalian display cell culture protocol

[0187] All 293t cells were grown in high glucose DMEM supplemented with Glutamax and pyruvate, 10% fetal bovine serum, and 1% penicillin / streptomycin. Cells were transduced at an MOI of approximately 0.1. 24 hours after transduction, 2 pg / ml of puromycin was added to the cells, and cells were constantly grown in media containing 2 pg / ml puromycin for subsequentsteps. Once a sufficient number of cells had grown, cells were physically dislodged from the cell culture flask. They were then washed in PBS, followed by staining with monoclonal antibodies of interest for 15 minutes at RT. Cells were then washed again with PBS, following which they were stained with anti-Myc FITC (Invitrogen, 13-2511) and anti-human IgG BV421 (BD Biosciences, 562581) antibodies for 15 minutes at RT. Cells were washed twice, resuspended in PBS, and finally stained with 7-AAD (ThermoFisher, A1310) before sorting. The top 1-5% of iGL binding cells were collected by sorting on a BD FACS Melody sorter.Mammalian display sequencing

[0188] Genomic DNA was extracted from sorted cells using a GenElute mammalian genomic DNA miniprep kit (Millipore Sigma, G1N70). Forward and reverse primers containing the Illumina P5 and P7 adapters respectively were designed to target regions of the Q23.PDGFR construct outside the area of the scanning NNK library, and used to amplify the region of interest. Amplified libraries were then purified using AMPureXP beads, and checked on a BioAnalyzer instrument to confirm that amplicons were of the correct size. The libraries were then sequenced on an Illumina MiSeq instrument using a 600 cycle MiSeq v3 kit.SHIV infection of rhesus macaques

[0189] Three days prior to SHIV infection, rhesus macaques were subcutaneously injected with 25mg / kg anti-CD8a mAb (MT807R1). Macaques were inoculated with SHIV by intravenous infusion. Blood draws, processing, and storage was performed as previously described (Roark et al., 2021, Science, 371, eabd2638).Env single genome sequencing

[0190] 3’ SHIV half genomes were sequenced as previously described (Li et al., 2016,Proc Natl Acad Sci U S A 113, E3413-22). Briefly, viral RNA was synthesized from plasma virions using the Qiagen BioRobot EZ1 Workstation with EZ1 Virus Mini Kit v2.0 (Qiagen). Viral RNA was then used to synthesize cDNA using SuperScript III reverse transcriptase (Invitrogen). cDNA was then serially diluted in 96 well plates and amplified using nested PCR such that <30% of wells were PCR-positive. Positive wells were sequenced using an Illumina Miseq sequencer.EXAMPLE 2: HIV Q23 Env trimer CD4bs

[0191] HIV Q23 trimers were designed that have mutations in the CD4bs epitope. These mutations help guide VRC01 -class and VH1-46 class antibody lineages toward becoming bnAbs.

[0192] Figure 17 depicts Q23 mutants by computational design.

[0193] Figure 18 depicts PTs-ELISA binding of Q23 CD4bs mutants.

[0194] Figure 19 depicts the mammalian display strategy.

[0195] Figure 20 depicts NNK scanning region of site-saturation library.

[0196] Figure 21 depicts the results of sorting against CD4bs and bnAbs and their intermediates.

[0197] Figure 22 depicts the conformation of mutations derived from mammalian display.

[0198] Figure 23 depicts the results of directed evolution by mammalian display to guide Q23-based immunogen designs for VRC01 -class early intermediates. Selected GT8 elicited antibodies from trail I are shown.

[0199] Figure 24 depicts strong binding mutants against GT8 elicited antibodies from trail I.

[0200] Figure 25 depicts combination mutant enhanced binding to early intermediates of VRC01 -Class.

[0201] Figure 26 depicts NS-EMPEM of the polyclonal antibody responses to Q23.V033GT in rhesus macaques CK11 and CK52.EXAMPLE 3: SEQUENCESTABLE 1: SEQUENCE SUMMARY

[0202] Some of the sequences include an IgE leader sequence (MDWTWILFLVAAATRVHS; SEQ ID NO:289). The invention also encompasses these sequences without an IgE leader sequence or without a sequence encoding the IgE leader sequence (SEQ ID NO:290).

[0203] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0204] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation thoserelating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMSWhat is claimed:

1. A polypeptide comprising an amino acid sequence encoding a variant HIV envelope protein.

2. The polypeptide of claim 1, wherein said amino acid sequence comprises one or more selected from the group consisting of: a) an amino acid sequence selected from the group consisting of: SEQ ID NO:1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190; b) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190; c) an amino acid sequence comprising at least 70% of the length of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190; and d) an amino acid sequence at least 90% identical to and comprising at least 70% the full length of an amino acid sequence selected from the group consisting of: SEQ ID NO:1-98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190.

3. The polypeptide of claim 2, wherein said polypeptide further comprises a nanoparticle scaffold.

4. The polypeptide of claim 1, wherein the amino acid sequence is encoded by a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence selected from the group consisting of: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, and SEQ ID NO: 191-288; b) a nucleotide sequence at least 90% identical to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 and SEQ ID NO: 191-288; c) a nucleotide sequence comprising at least 70% of the full length of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, and SEQ ID NO: 191-288; and d) a nucleotide sequence at least 90% identical to and comprising at least 70% the full length of a nucleotide sequence selected from the group consisting of: SEQ ID NO: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 and SEQ ID NO: 191-288.

5. An immunogenic composition comprising a polypeptide of any one of claims6. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of any one of claims 1-4.

7. An immunogenic composition, comprising a nucleic acid molecule of claim 6.

8. The immunogenic composition of claim 7 comprising a nanoparticle comprising the nucleic acid molecule.

9. A method of inducing an immune response against HIV in a subject in need thereof, the method comprising administering to the subject the polypeptide of any one of claims 1-4, the composition of claim 5, the nucleic acid molecule of claim 6 or the composition of any one of claim 7 or 8.

10. The method of claim 9, wherein said subject is a human.

11. The method of claim 9, wherein the subject is infected with HIV or at risk of becoming infected with HIV.

12. A method of treating HIV infection in a subject in need thereof, comprising administering to the subject the polypeptide of any one of claims 1-4, the composition of claim 5, the nucleic acid molecule of claim 6 or the composition of any one of claim 7 or 8.

13. The method of claim 12, wherein said subject is a human.

14. A method of preventing development of a disease or disorder associated with HIV infection in a subject in need thereof, comprising administering to the subject the polypeptide of any one of claims 1-4, the composition of claim 5, the nucleic acid molecule of claim 6 or the composition of any one of claim 7 or 8.

15. The method of claim 14, wherein the subject is infected with HIV or at risk of becoming infected with HIV.

16. The method of claim 14, wherein the disease or disorder associated with HIV infection comprises AIDS.

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