HIV-1 ENV immunogens that target UCAS against multiple antigenic sites

Abstract

The invention is directed to modified HIV-1 envelopes, compositions comprising these modified envelopes, nucleic acids encoding these modified envelopes, compositions comprising these nucleic acids, and methods of using these modified HIV-1 envelopes and / or these nucleic acids to induce immune responses.

Description

Attorney Docket: 2933311.091.WO1 DU8448PCT HIV-1 ENV IMMUNOGENS THAT TARGET UCAS AGAINST MULTIPLE ANTIGENIC SITES

[0001] This International Patent Application claims the benefit of and priority to U.S. Application No.63 / 634,195, filed April 15, 2024, entitled “HIV-1 ENV IMMUNOGENS THAT TARGET UCAS AGAINST MULTIPLE ANTIGENIC SITES,” the content of which is hereby incorporated by reference in its entirety. STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant UM1-AI144371 awarded by the NIH, NIAID, Division of AIDS and HHS. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 1, 2025, is named 2933311_091PCT.xml and is 42,122 bytes in size. TECHNICAL FIELD

[0004] The present invention relates in general, to a composition suitable for use in inducing anti-HIV-1 antibodies, and, in particular, to immunogenic compositions comprising envelope proteins and nucleic acids to induce cross-reactive neutralizing antibodies and increase their breadth of coverage. The invention also relates to methods of inducing such broadly neutralizing anti-HIV-1 antibodies using such compositions. BACKGROUND OF THE INVENTION

[0005] The development of a safe and effective HIV-1 vaccine is one of the highest priorities of the scientific community working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients, ART is not routinely available in developing countries.Attorney Docket: 2933311.091.WO1 DU8448PCT SUMMARY OF THE INVENTION

[0006] Forty years of HIV-1 vaccine design have been largely ineffective, prompting the development of extensive sequential vaccination regimens that are intended to mimic the antigenic diversity of the HIV-1 Envelope (Env) as it co-evolves in response to the humoral immune system (1, 2). Such sequential vaccination strategies are predicated on the existence of immunogens which are capable of first engaging broadly neutralizing antibody (bnAb) precursors to stimulate their proliferation and development into mature bnAbs. These precursors, or unmutated common ancestors (UCAs), are notoriously difficult to stimulate via vaccination, in large part due to their low frequency within the human B cell repertoire. However, extensive research has resulted in steady progress being made towards the development of UCA-targeting immunogens. Generally, these immunogens are based on HIV-1 Env sequences isolated from people living with HIV who have naturally developed bnAbs in response to infection. These native sequences are then modified through rational design or random mutagenesis to facilitate binding to UCAs. One example of such an immunogen is CH505 M5.G458Y, which has been engineered to bind to the UCA from the CD4 binding site (CD4bs)-directed CH235 lineage (3, 4). Another such UCA-targeting immunogen is the V1swap Env, which is predominantly derived from the CH84810.17 Env (5), but has subjected to structure-based design and modification through high-throughput mammalian cell display to render it capable of engaging the UCAs from multiple distinct V3 glycan bnAb lineages, including DH270, BG18 and BF520.1 (6-9). Similar work has been pursued to develop immunogens which are capable of binding to UCAs that can go on to become V2 apex bnAbs. CAP256, ZM233 and BG505-derived Envelopes have all shown some preliminary promise at being able to select for V2 precursors with the extremely long CDRH3s that are thought to be required to form quaternary contacts at the apex of the Env trimer (10-12).

[0007] Despite these many advances, existing UCA-targeting Env immunogens have been focused solely on stimulating the development of bnAbs against individual antigenic domains. In order to prime an immune response which could go on to generate polyclonal bnAbs against multiple conserved epitopes, here we describe the development of chimeric Envelopes which are capable of binding to UCAs from multiple bnAb epitopes simultaneously (Figure 1).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0008] In certain embodiments, the invention provides compositions and methods for induction of an immune response, for example cross-reactive (broadly) neutralizing (bn) Ab induction.

[0009] In certain aspects the invention provides a recombinant protein or nucleic acid encoding a recombinant protein as described in SEQ ID NOS: 1-10, and 21-25. In certain aspects the invention provides a selection of HIV-1 envelopes for use as prime and boost immunogens in methods to induce HIV-1 neutralizing antibodies. In certain aspects, the invention provides a selection of HIV-1 envelopes for use as a boost immunogen in methods to induce HIV-1 neutralizing antibodies. In certain aspects, the invention provides a selection of HIV-1 envelopes for use as a prime immunogen in methods to induce HIV-1 neutralizing antibodies.

[0010] In certain embodiments, the invention provides a recombinant HIV-1 envelope protein or nucleic acid encoding a recombinant HIV-1 envelope protein as described in SEQ ID NOS: 1-10, and 21-24.

[0011] In certain embodiments, the compositions contemplate nucleic acid, as DNA and / or RNA, or protein immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and / or RNA, immunization either alone or in combination with envelope protein(s).

[0012] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0013] In certain aspects the invention provides a composition comprising at least one nucleic acid encoding an HIV-1 envelope of the invention.

[0014] In certain embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide instead of a nucleic acid sequence encoding the HIV-1 envelope. In certain embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide, a nucleic acid sequence encoding the HIV-1 envelope, or a combination thereof. In certain embodiments, the polypeptides are recombinantly produced.

[0015] The envelope used in the compositions and methods of the invention can be a gp160, gp150, gp145, gp140, gp120, gp41, or N-terminal deletion variants thereof asAttorney Docket: 2933311.091.WO1 DU8448PCT described herein, cleavage resistant variants thereof as described herein, or codon optimized sequences thereof. In certain embodiments the composition comprises envelopes as trimers. In certain embodiments, envelope proteins are multimerized, for example trimers are attached to a particle such that multiple copies of the trimer are attached and the multimerized envelope is prepared and formulated for immunization in a human. In certain embodiments, the compositions comprise envelopes, including but not limited to trimers as particulate, high-density array on liposomes or other particles, for example but not limited to nanoparticles. In some embodiments, the trimers are in a well ordered, near native like or closed conformation. In some embodiments the trimer compositions comprise a homogenous mix of native like trimers. In some embodiments the trimer compositions comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 95% native like trimers.

[0016] The polypeptide contemplated by the invention can be a polypeptide comprising any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting essentially of any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting of any one of the polypeptides described herein. In certain embodiments, the polypeptide is recombinantly produced. In certain embodiments, the polypeptides and nucleic acids of the invention are suitable for use as an immunogen, for example to be administered in a human subject.

[0017] In certain embodiments, the envelopes of the present invention are designed to form a stable trimer. In certain embodiments envelope protomers form a trimer which is not a SOSIP timer. In certain embodiment the trimer is a SOSIP based trimer wherein each protomer comprises additional modifications. In certain embodiments, envelope trimers are recombinantly produced. In certain embodiments, envelope trimers are purified from cellular recombinant fractions by antibody binding and reconstituted in lipid comprising formulations. See for example WO2015 / 127108 titled “Trimeric HIV-1 envelopes and uses thereof” which content is herein incorporated by reference in its entirety. In certain embodiments, the envelopes of the invention are engineered and comprise non-naturally occurring modifications.

[0018] In certain embodiments, the envelope is in a liposome. In certain embodiments the envelope comprises a transmembrane domain with a cytoplasmic tail embedded in a liposome. In certain embodiments, the nucleic acid comprises a nucleic acid sequence whichAttorney Docket: 2933311.091.WO1 DU8448PCT encodes a gp120, gp140, gp145, gp150, or gp160. In certain embodiments, where the nucleic acids are operably linked to a promoter and inserted in a vector, the vectors are any suitable vector. Non-limiting examples include, VSV, replicating rAdenovirus type 4, MVA, Chimp adenovirus vectors, pox vectors, and the like. In certain embodiments, the nucleic acids are administered in NanoTaxi block polymer nanospheres. In certain embodiments, the compositions and methods comprise an adjuvant. Non-limiting examples include, AS01 B, AS01 E, gla / SE, alum, Poly I poly C (poly IC), polyIC / long chain (LC) TLR agonists, TLR7 / 8 and 9 agonists, or a combination of TLR7 / 8 and TLR9 agonists (see Moody et al. (2014) J. Virol. March 2014 vol.88 no.63329-3339), or any other adjuvant. Non-limiting examples of TLR7 / 8 agonist include TLR7 / 8 ligands, Gardiquimod, Imiquimod and R848 (resiquimod). A non-limiting embodiment of a combination of TLR7 / 8 and TLR9 agonist comprises R848 and oCpG in STS (see Moody et al. (2014) J. Virol. March 2014 vol.88 no. 63329-3339).

[0019] In non-limiting embodiments, the adjuvant is a lipid nanoparticle (LNP). See e.g., without limitation Shirai et al. “Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses” Vaccines 2020, 8, 433; doi:10.3390 / vaccines8030433, published 3 August 2020. In non-limiting embodiments, LNPs used as adjuvants for protein compositions are composed of an ionizable lipid, cholesterol, lipid conjugated with polyethylene glycol, and a helper lipid. Non-limiting embodiment include LNPs without polyethylene glycol.

[0020] In certain aspects the invention provides a cell comprising a nucleic acid encoding any one of the envelopes of the invention suitable for recombinant expression. In certain aspects, the invention provides a clonally derived population of cells encoding any one of the envelopes of the invention suitable for recombinant expression. In certain aspects, the invention provides a stable pool of cells encoding any one of the envelopes of the invention suitable for recombinant expression.

[0021] In certain aspects the invention provides nucleic acids encoding HIV-1 envelopes for immunization wherein the nucleic acid encodes a gp120 envelope, gp120D8 envelope, a gp140 envelope (gp140C, gp140CF, gp140CFI) as soluble or stabilized protomer of a SOSIP trimer, a gp145 envelope, a gp150 envelope, or a transmembrane bound envelope.

[0022] In certain embodiments, the compositions for use in immunization further comprise an adjuvant.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0023] In certain embodiments, wherein the compositions comprise a nucleic acid, the nucleic acid is operably linked to a promoter, and could be inserted in an expression vector.

[0024] In one aspect the invention provides a composition for a prime boost immunization regimen comprising any of envelopes described herein, or any combination thereof wherein the envelope is a prime or boost immunogen. In certain embodiments, the composition for a prime boost immunization regimen comprises one or more envelopes described herein, wherein the polypeptide is a non-naturally occurring protomer designed to form an envelope trimer, wherein the envelope is a prime or boost immunogen. In one aspect the invention provides a composition for a prime boost immunization regimen comprising one or more envelopes of the invention. In one aspect the invention provides a composition for a prime immunization comprising one or more envelopes of the invention.

[0025] In certain aspects the invention provides methods of inducing an immune response in a subject comprising administering a composition comprising a polypeptide and / or any suitable form of a nucleic acid(s) encoding an HIV-1 envelope(s) in an amount sufficient to induce an immune response.

[0026] In certain aspects, the invention provides a pharmaceutical composition comprising any one of the recombinant trimers of the invention. In certain embodiments the compositions comprising trimers are immunogenic. The percent trimer in such immunogenic compositions could vary. In some embodiments the composition comprises 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% stabilized trimer.

[0027] In certain embodiments, the recombinant protein nanoparticle comprises the envelope and ferritin. In certain embodiments, the inventive designs comprise modifications, including without limitation linkers between the envelope and ferritin designed to optimize ferritin nanoparticle assembly.

[0028] In certain aspects, the invention provides a composition comprising any one of the inventive envelopes or nucleic acid sequences encoding the same.

[0029] In certain aspects, the invention provides compositions comprising a nanoparticle which comprises any one of the envelopes of the invention.

[0030] In certain embodiments, the nanoparticle is a ferritin self-assembling nanoparticle.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0031] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the envelopes of the invention. In certain embodiments, the composition is administered as a prime and / or a boost. In certain embodiments, the composition comprises nanoparticles. In certain embodiments, methods of the invention further comprise administering an adjuvant.

[0032] In certain aspects, the invention provides a composition comprising a plurality of nanoparticles comprising a plurality of the envelopes / trimers of the invention. In non- limiting embodiments, the envelopes / trimers of the invention are multimeric when comprised in a nanoparticle. The nanoparticle size is suitable for delivery. In non-liming embodiments the nanoparticles are ferritin-based nanoparticles.

[0033] In certain aspects, the invention provides nucleic acids comprising sequences encoding polypeptides or proteins of the invention. In certain embodiments, the nucleic acids are DNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0034] In some aspects, the invention provides a recombinant trimer comprising three identical protomers of an envelope from SEQ ID NOS: 1-5 and 21-24. In some embodiments, the invention provides an immunogenic composition comprising the recombinant trimer and a carrier, wherein the trimer comprises three identical protomers of an HIV-1 envelope listed in SEQ ID NOS: 1-5 and 21-24.

[0035] In some embodiments, the invention provides an immunogenic composition comprising a nucleic acid encoding the recombinant HIV-1 envelope and a carrier. In some embodiments, the envelopes are or are designed as trimers, and / or nanoparticles.

[0036] In some aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises an envelope, wherein the envelope is selected from SEQ ID NOS: 1-10 and 21-25, or any combination thereof. In some embodiments, the compositions comprise two, three, four or more different immunogens. In some embodiments the immunogens target different CH235 lineage members. In some embodiments, the immunogens target the CH235 lineage UCA. In non-limiting embodiments the different immunogens are selected from the various envelope designs described herein.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0037] In some embodiments, the nanoparticle of the composition is a ferritin self- assembling nanoparticle.

[0038] In some aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises a nucleic acid encoding the recombinant HIV-1 envelope polypeptide from SEQ ID NOS: 1-5 and 21-24.

[0039] In some embodiments, the nanoparticle of the composition comprises multimers of trimers.

[0040] In some embodiments, the nanoparticle of the composition comprises 1-8 trimers.

[0041] In some aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant envelopes or compositions described herein. In some embodiments the methods comprise administering two, three, four or more different immunogens. In some embodiments, the different immunogens target different CH235 lineage members. In some embodiments, the different immunogens target the CH235 lineage UCA or CH235 intermediate antibodies. In non-limiting embodiments the different immunogens are selected from the envelope designs described herein at SEQ ID NOS: 1-5 and 21-24.

[0042] In certain embodiments, the subject is infected with HIV (e.g., HIV-1). In certain embodiments, the subject is an HIV-uninfected individual. In certain embodiments, the subject is an HIV-infected individual. In certain embodiments, the administration to the HIV-infected individual induces broadly neutralizing antibodies. In certain embodiments the broadly neutralizing antibodies of the HIV-infected individual mediates viral (e.g., HIV-1) clearance from blood and tissues.

[0043] In some embodiments, the composition is administered as a single prime or as repetitive immunization prime. In preferred embodiments, the repetitive immunization is administered 3 or 4 times.

[0044] In some embodiments, the composition is administered as a single boost or as a repetitive series of boosts. In preferred embodiments, the repetitive series of boosts is administered 3 or 4 times.

[0045] In some embodiments, the composition is a first composition administered as a prime. In some embodiments, the composition is a second composition administered as one or more boosts. In some embodiments, the method comprises administering the firstAttorney Docket: 2933311.091.WO1 DU8448PCT composition as a prime and administering the second composition as one or more boosts. In preferred embodiments, the first composition and the second composition are different.

[0046] In some aspects, the invention provides a nucleic acid encoding any of the recombinant envelopes described herein. In some embodiments, the invention provides a composition comprising the nucleic acid and a carrier.

[0047] In some embodiments, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising the nucleic acid encoding any of the recombinant envelopes described herein. In some embodiments, the immunogenic composition further comprises a carrier.

[0048] In certain aspects, the invention provides an immunogenic composition or composition, wherein the composition comprises at least two different HIV-1 envelope polypeptides or nucleic acids encoding a recombinant HIV-1 envelope polypeptide, or a combination thereof.

[0049] In certain aspects, the invention provides an immunogenic composition comprising a first immunogen and a second immunogen, wherein the first immunogen is a recombinant HIV-1 envelope polypeptide from Table 1, or encoded by a nucleic acid encoding said recombinant HIV-1 envelope polypeptide, and wherein the second immunogen is a different recombinant HIV-1 envelope polypeptide from Table 1 or a nucleic acid encoding said different recombinant HIV-1 envelope polypeptide. In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering the immunogenic composition in an amount sufficient to induce an immune response. In certain embodiments, the method further comprises administering an agent which modulates host immune tolerance.

[0050] In certain embodiments, at least one of the first immunogen and the second immunogen is a recombinant HIV-1 envelope polypeptide. In certain embodiments, at least one of the first immunogen and the second immunogen is a recombinant trimer comprising three identical protomers of the recombinant HIV-1 envelope polypeptide. In certain embodiments, the first immunogen and the second immunogen are a recombinant HIV-1 envelope polypeptide. In certain embodiments, at least one of the first immunogen and the second immunogen is a nucleic acid. In certain embodiments, the first immunogen and the second immunogen are a nucleic acid.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0051] In certain embodiments, the HIV-1 envelopes are in the form of a recombinant HIV-1 envelope polypeptides or nucleic acid, or a combination thereof. In certain embodiments, one or more of the HIV-1 envelopes is a recombinant trimer comprising three identical protomers of the recombinant HIV-1 envelope polypeptide. In certain embodiments, the composition comprises a carrier. In certain embodiments, the composition further comprises an adjuvant. BRIEF DESCRIPTION OF THE FIGURES

[0052] Figure 1 shows a schematic of the design rationale for generating a universal HIV Env priming immunogen. The prefusion structures of three classes of well-characterized UCA targeting Env immunogens are shown: V1swap Env (green) (Figure 1, left) is derived from CH84810.17 but has undergone extensive modification of its V3 glycan epitope and proximal domains in order to facilitate the binding of multiple V3 glycan UCAs. Immunogens derived from the CH505 Env (orange) (Figure 1, center) have been shown to bind with high affinity to UCAs that can go on to mature into CD4 binding site (CD4bs) bnAbs. Similarly, several Env candidates have been proposed as priming immunogens to target V2 apex UCAs, including CAP256 (blue) (Figure 1, right). In an effort to combine the favorable antigenic characteristics from each of these classes of immunogens, chimeric Envelopes are under development that make use of a CH505 “backbone” onto which a variety of V3 glycan and V2 apex modifications can be grafted.

[0053] Figures 2A-2B shows expression and antigenic characterization of soluble CH505pUCA ectodomain. (2A) Size-exclusion chromatogram of the CH505pUCA Env after PGT145 affinity chromatography. The profile from this Superose6 Increase 10-300 column shows a homogeneous peak at the expected elution volume for a densely glycosylated prefusion trimer. (2B) Antigenicity of CH505pUCA Env evaluated by biolayer interferometry (BLI). His-tagged, soluble CH505pUCA Env ectodomain was immobilized to BLI NiNTA sensortips before being dipped into the listed monoclonal antibodies (mAbs). Although the CH505pUCA Env has not yet been modified to enhance targeting of V2 apex UCAs, it still exhibits detectable binding to the reverted unmutated ancestor (RUA) of the V2-directed bnAb CH01 (green).

[0054] Figure 3 shows expression levels of transmembrane-immobilized CH505pUCA constructs by flow cytometry. Mean fluorescence intensities (MFI) are plotted for each cellAttorney Docket: 2933311.091.WO1 DU8448PCT population after being stained with the mAb listed above each plot. Cell populations were transfected with the corresponding construct listed below on the x-axis. Mock cells (black) were untransfected and serve as a negative control.

[0055] Figure 4 shows antigenicity of transmembrane CH505pUCA Envelope. Cells transfected with the CH505pUCA gp150755 construct (DW-198) were evaluated for binding to a panel of 24 mAbs which are listed on the x-axis. The y-axis plots MFI after staining with each mAb. Antibodies are colored based on their respective binding characteristics, with CD4 binding site UCAs colored orange, V3 glycan UCAs colored green, V2 apex UCAs colored blue, broadly neutralizing antibodies (bnAbs) colored purple and base-binders and non- neutralizing antibodies (nonAbs) colored yellow. The influenza HA-directed mAb CH65 is colored black and was included as a negative control.

[0056] Figures 5A-5B shows the expression and antigenic characterization of soluble CH505pUCAI130H CstrandOPT ectodomain. (5A) Size-exclusion chromatogram of the CH505pUCA I130H CstrandOPT Env after PGT145 affinity chromatography. The profile from this Superose6 Increase 10-300 column shows a homogeneous peak at the expected elution volume for a densely glycosylated prefusion trimer. (5B) Antigenicity of CH505pUCA I130H CstrandOPT Env evaluated by biolayer interferometry (BLI). His- tagged, soluble CH505pUCA I130H CstrandOPT Env ectodomain was immobilized to BLI NiNTA sensortips before being dipped into the listed monoclonal antibodies (mAbs).

[0057] Figures 6A-6B: Antigenicity of transmembrane CH505pUCA I130H CstrandOPT Envelopes. (6A) Cells transfected with mRNA-encoded, transmembrane- immobilized Env constructs were evaluated for binding to the panel of 24 antibodies identified in each graph. The mRNA constructs are listed in panel (6B).

[0058] Figure 7: The amino acid sequence of DW-301: CH505pUCA_I130H CstrandOPT SOSIP-2P (AA) (SEQ ID NO: 21) is shown. Blue highlighting corresponds to I130H and CstrandOPT substitutions, yellow highlighting denotes N197D substitution and green highlighting corresponds to V1swap-derived substitutions. DETAILED DESCRIPTION OF THE INVENTION

[0059] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the termsAttorney Docket: 2933311.091.WO1 DU8448PCT used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0060] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0061] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0062] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0063] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0064] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0065] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. HIV Recombinant Envelopes

[0066] The development of a safe, highly efficacious prophylactic HIV-1 vaccine is of paramount importance for the control and prevention of HIV-1 infection. A major goal ofAttorney Docket: 2933311.091.WO1 DU8448PCT HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) (Immunol. Rev.254: 225-244, 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not reproducibly induced by current vaccines.

[0067] For the past 25 years, the HIV vaccine development field has used single or prime boost heterologous Envs as immunogens, but to date has not found a regimen to induce high levels of bnAbs.

[0068] Recently, a new paradigm for design of strategies for induction of broadly neutralizing antibodies was introduced, that of B cell lineage immunogen design (Nature Biotech.30: 423, 2012) in which the induction of bnAb lineages is recreated. It recently demonstrated the power of mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction (Nature 496: 469, 2013).

[0069] Described herein are nucleic and amino acids sequences of HIV-1 envelopes. The sequences for use as immunogens are in any suitable form.

[0070] An HIV-1 envelope has various structurally defined fragments / forms: gp160; gp140, including cleaved gp140 and uncleaved gp140 (gp140C), gp140CF, or gp140CFI; gp120 and gp41. A skilled artisan appreciates that these fragments / forms are defined not necessarily by their crystal structure, but by their design and bounds within the full length of the gp160 envelope. While the specific consecutive amino acid sequences of envelopes from different strains are different, the bounds and design of these forms are well known and characterized in the art.

[0071] For example, it is well known in the art that during its transport to the cell surface, the gp160 polypeptide is processed and proteolytically cleaved to gp120 and gp41 proteins. Cleavages of gp160 to gp120 and gp41 occurs at a conserved cleavage site “REKR” (SEQ ID NO: 11). See Chakrabarti et al. Journal of Virology vol.76, pp.5357- 5368 (2002) see for example Figure 1, and second paragraph in the Introduction on p.5357; Binley et al. Journal of Virology vol.76, pp.2606-2616 (2002) for example at Abstract; Gao et al. Journal of Virology vol.79, pp.1154-1163 (2005); Liao et al. Virology vol.353(2): 268–282 (2006).

[0072] The role of the furin cleavage site was well understood both in terms of improving cleave efficiency, see Binley et al. supra, and eliminating cleavage, see Bosch and Pawlita, Virology 64 (5):2337-2344 (1990); Guo et al. Virology 174: 217-224 (1990); McCune et al. Cell 53:55-67 (1988); Liao et al. J Virol. Apr;87(8):4185-201 (2013).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0073] Likewise, the design of gp140 envelope forms is also well known in the art, along with the various specific changes which give rise to the gp140C (uncleaved envelope), gp140CF and gp140CFI forms. Envelope gp140 forms are designed by introducing a stop codon within the gp41 sequence. See Chakrabarti et al. at Figure 1.

[0074] Envelope gp140C refers to a gp140 HIV-1 envelope design with a functional deletion of the cleavage (C) site, so that the gp140 envelope is not cleaved at the furin cleavage site. The specification describes cleaved and uncleaved forms, and various furin cleavage site modifications that prevent envelope cleavage are known in the art. In some embodiments of the gp140C form, two of the R residues in and near the furin cleavage site are changed to E, e.g., RRVVEREKR (SEQ ID NO: 12) is changed to ERVVEREKE (SEQ ID NO: 13), and is one example of an uncleaved gp140 form. Another example is the gp140C form which has the REKR site (SEQ ID NO: 11) changed to SEKS (SEQ ID NO: 14). See supra for references.

[0075] Envelope gp140CF refers to a gp140 HIV-1 envelope design with a deletion of the cleavage (C) site and fusion (F) region. Envelope gp140CFI refers to a gp140 HIV-1 envelope design with a deletion of the cleavage (C) site, fusion (F) and immunodominant (I) region in gp41. See Chakrabarti et al. Journal of Virology vol.76, pp.5357-5368 (2002) see for example Figure 1, and Second paragraph in the Introduction on p.5357; Binley et al. Journal of Virology vol.76, pp.2606-2616 (2002) for example at Abstract; Gao et al. Journal of Virology vol.79, pp.1154-1163 (2005); Liao et al. Virology vol.353(2): 268–282 (2006).

[0076] In other embodiments, the delta N-design described for CH505 T / F envelope can be used to make delta N-designs of other CH505 envelopes. In certain embodiments, the invention relates generally to an immunogen, gp160, gp120 or gp140, without an N-terminal Herpes Simplex gD tag substituted for amino acids of the N-terminus of gp120, with an HIV leader sequence (or other leader sequence), and without the original about 4 to about 25, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids of the N-terminus of the envelope (e.g. gp120). See US Patent 10,040,826, e.g. at pages 10-12, the contents of which is hereby incorporated by reference in its entirety.

[0077] The general strategy of deletion of N-terminal amino acids of envelopes results in proteins, for example gpl20s, expressed in mammalian cells that are primarily monomeric, as opposed to dimeric, and, therefore, solves the production and scalability problem ofAttorney Docket: 2933311.091.WO1 DU8448PCT commercial gp120 Env vaccine production. In other embodiments, the amino acid deletions at the N-terminus result in increased immunogenicity of the envelopes.

[0078] In some embodiments, the HIV envelopes described herein include the amino acid sequence set forth in any one of SEQ ID NOs: 1-5 and 21-24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NOs: 1-5 and 21-24.

[0079] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0080] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0081] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0082] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0083] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0084] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0085] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%,Attorney Docket: 2933311.091.WO1 DU8448PCT 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0086] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0087] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0088] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of any one of SEQ ID NOs: 1-5 and 21-24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-5 and 21-24.

[0089] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0090] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0091] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0092] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0093] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%,Attorney Docket: 2933311.091.WO1 DU8448PCT 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0094] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0095] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0096] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0097] In some embodiments, the HIV envelope includes all the amino acids after the signal peptide of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0098] In some embodiments, the HIV envelopes describe herein include the nucleotide sequence set forth in any one of SEQ ID NO: 6-10 and 25, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one nucleotide sequences SEQ ID NO: 6-10 and 25.

[0099] In some embodiments, the HIV envelope includes the nucleotide sequence set forth in SEQ ID NO: 6 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6.

[0100] In some embodiments, the HIV envelope includes the nucleotide sequence set forth in SEQ ID NO: 7 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7.

[0101] In some embodiments, the HIV envelope includes the nucleotide sequence set forth in SEQ ID NO: 8 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%,Attorney Docket: 2933311.091.WO1 DU8448PCT 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8.

[0102] In some embodiments, the HIV envelope includes the nucleotide sequence set forth in SEQ ID NO: 9 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 9.

[0103] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0104] In some embodiments, the HIV envelope includes the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25.

[0105] Table 1 HIV recombinant envelope SEQUENCEAttorney Docket: 2933311.091.WO1 DU8448PCT

[0106] In some embodiments, the HIV-1 recombinant envelopes described herein bind to neutralizing antibodies. In some embodiments, any of the HIV recombinant envelopes described (e.g. Table 1) can recognize and / or bind to any of HIV neutralizing antibodies 2G12, N6, PGT145, DH270.6, PGT151, CH235.12, or a combination thereof.

[0107] In some embodiments, any of the HIV recombinant envelopes included in Table 1 can bind to 2G12.

[0108] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to 2G12.

[0109] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to 2G12.

[0110] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to 2G12.

[0111] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to 2G12.

[0112] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to 2G12.

[0113] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to N6.

[0114] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to N6.

[0115] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to N6.

[0116] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to N6.

[0117] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to N6.

[0118] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to PGT145.

[0119] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to PGT145.

[0120] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to PGT145.

[0121] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to PGT145.

[0122] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to PGT145.

[0123] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to DH270.6.

[0124] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to DH270.6.

[0125] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to DH270.6.

[0126] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to DH270.6.

[0127] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to DH270.6.

[0128] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to PGT151.

[0129] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to PGT151.

[0130] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to PGT151.

[0131] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to PGT151.

[0132] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to PGT151.

[0133] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to CH235.12.

[0134] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to CH235.12.

[0135] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to CH235.12.

[0136] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to CH235.12.

[0137] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to CH235.12.

[0138] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to CH235.12.

[0139] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to CH235.12.

[0140] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to CH235.12.

[0141] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to CH235.12.

[0142] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to BG18.

[0143] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to BG18.

[0144] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to BG18.

[0145] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to BG18.

[0146] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to DH270.

[0147] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to DH270.

[0148] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to DH270.

[0149] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to DH270.

[0150] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to 2G12.

[0151] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to 2G12.

[0152] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to 2G12.

[0153] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to 2G12.

[0154] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to PGT128.

[0155] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to PGT128.

[0156] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to PGT128.

[0157] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to PGT128.

[0158] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to N6.

[0159] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to N6.

[0160] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to N6.

[0161] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to N6.

[0162] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to CH235.12.

[0163] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to CH235.12.

[0164] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to CH235.12.

[0165] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to CH235.12.

[0166] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to PGT151.

[0167] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to PGT151.

[0168] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to PGT151.

[0169] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to PGT151.

[0170] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to DH511.

[0171] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to DH511.

[0172] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to DH511.

[0173] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to DH511.

[0174] In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize gp120. In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize gp140. In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize gp160.

[0175] In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize the V3-glycan site. In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize the CD4 binding site. In some embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize the V2 apex site.

[0176] In some embodiments, the HIV recombinant envelopes described herein can bind to broadly neutralizing antibodies (bnAbs). In some embodiments, the HIV recombinant envelopes described herein can bind to base-binders and non-neutralizing antibodies (nonAbs).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0177] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0178] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0179] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0180] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0181] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0182] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0183] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0184] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0185] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to an antibody that recognizes / bind to a CD4 binding site UCA.

[0186] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0187] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0188] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0189] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0190] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0191] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0192] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to an antibody that recognizes / bind to a V3 glycan UCA.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0193] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0194] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to an antibody that recognizes / bind to a V3 glycan UCA.

[0195] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0196] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0197] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0198] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0199] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0200] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to an antibody that recognizes / bind to a V2 apex UCA.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0201] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0202] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0203] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to an antibody that recognizes / bind to a V2 apex UCA.

[0204] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0205] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0206] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0207] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0208] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0209] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0210] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0211] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0212] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to an antibody that recognizes / bind to HIV broadly neutralizing antibodies (bnAbs).

[0213] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0214] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%,Attorney Docket: 2933311.091.WO1 DU8448PCT 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0215] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0216] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0217] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0218] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0219] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0220] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequenceAttorney Docket: 2933311.091.WO1 DU8448PCT of SEQ ID NO: 23 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs).

[0221] In some embodiments, the HIV recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can bind to an antibody that recognizes / bind to base-binders and non- neutralizing antibodies (nonAbs). Trimers

[0222] HIV-1 envelope trimers and other envelope designs

[0223] Stabilized HIV-1 Env trimer immunogens show enhanced antigenicity for broadly neutralizing antibodies and are not recognized by non-neutralizing antibodies. Envelope modifications and designs include, but are not limited to, trimers that are further multimerized, and / or used as particulate, high-density array in liposomes or other particles, for example but not limited to nanoparticles. Any one of the envelopes of the invention could be designed and expressed as described herein.

[0224] A stabilized chimeric SOSIP design can be used to generate CH505 trimers. This design is applicable to diverse viruses from multiple clades. SOSIP designs can be applied to the envelopes disclosed herein including those in Figure 1.

[0225] Elicitation of neutralizing antibodies is one goal for antibody-based vaccines. Neutralizing antibodies target the native trimeric HIV-1 Env on the surface virions. The trimeric HIV-1 envelope protein consists of three protomers each containing a gp120 and gp41 heterodimer. Recent immunogen design efforts have generated soluble near-native mimics of the Env trimer that bind to neutralizing antibodies but not non-neutralizing antibodies. The recapitulation of the native trimer could be a key component of vaccine induction of neutralizing antibodies. Neutralizing Abs target the native trimeric HIV-1 Env on the surface of viruses (Poignard et al. J Virol.2003 Jan;77(1):353-65; Parren et al. J Virol. 1998 Dec;72(12):10270-4.; Yang et al. J Virol.2006 Nov;80(22):11404-8.). The HIV-1 Env protein consists of three protomers of gp120 and gp41 heterodimers that are noncovalently linked together (Center et al. J Virol.2002 Aug;76(15):7863-7.). Soluble near-native trimers preferentially bind neutralizing antibodies as opposed to non-neutralizing antibodies (Sanders et al. PLoS Pathog.2013 Sep; 9(9): e1003618).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0226] Vaccination with immunogens that target bnAb B cell lineages and mimic the native trimers on virions may increase the frequency of broadly neutralizing plasma antibodies.

[0227] Previous work has shown that CH505 derived soluble trimers are hard to produce. From a study published by Julien et al in 2015 (Proc Natl Acad Sci U S A.2015 Sep 22; 112(38): 11947–11952.), it was shown that while CH505 produced comparable amounts of protein by transient transfection, only 5% of the CH505 protein formed trimer which 5 times lower than the gold standard viral strain BG505. Provided here are non- limiting embodiments of well-folded trimers for Env immunizations.

[0228] Near-native soluble trimers using the 6R.SOSIP.664 design are capable of generating autologous tier 2 neutralizing plasma antibodies in the plasma (Sanders et al. 2015), which provides a starting point for designing immunogens to elicit broadly neutralizing antibodies. While these trimers are preferentially antigenic for neutralizing antibodies, they still possess the ability to expose the V3 loop, which generally results in strain-specific binding and neutralizing antibodies after vaccination. Using the unliganded structure, the BG505.6R.SOSIP.664 has been stabilized by adding cysteines at position 201 and 433 to constrain the conformational flexibility such that the V3 loop is maintained unexposed (Kwon et al. Nat Struct Mol Biol.2015 Jul; 22(7): 522–531.).

[0229] Provided are engineered trimeric immunogens derived from multiple viruses from CH505. We generated chimeric 6R.SOSIP.664, chimeric disulfide stabilized (DS) 6R.SOSIP.664 (Kwon et al Nat Struct Mol Biol.2015 Jul; 22(7): 522–531.), chimeric 6R.SOSIP.664v4.1 (DeTaeye et al. Cell.2015 Dec 17;163(7):1702-15. doi: 10.1016 / j.cell.2015.11.056), and chimeric 6R.SOSIP.664v4.2 (DeTaeye et al. Cell.2015 Dec 17;163(7):1702-15. doi: 10.1016 / j.cell.2015.11.056). The 6R.SOSIP.664 is the basis for all of these designs and is made as a chimera of C.CH0505 and A.BG505. The gp120 of C.CH505 was fused with the BG505 inner domain gp120 sequence within the alpha helix 5 (^5) to result in the chimeric protein. The chimeric gp120 is disulfide linked to the A.BG505 gp41 as outlined by Sanders et al. (PLoS Pathog.2013 Sep; 9(9): e1003618). These immunogens were designed as chimeric proteins that possess the BG505 gp41 connected to the CH505 gp120, since the BG505 strain is particularly adept at forming well-folded, closed trimers. This envelope design retains the CH505 CD4 binding site that is targeted by the CH103 and CH235 broadly neutralizing antibody lineages that were isolated from CH505.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0230] Recombinant envelopes as trimers could be produced and purified by any suitable method. For a non-limiting example of purification methods see Ringe RP, Yasmeen A, Ozorowski G, Go EP, Pritchard LK, Guttman M, Ketas TA, Cottrell CA, Wilson IA, Sanders RW, Cupo A, Crispin M, Lee KK, Desaire H, Ward AB, Klasse PJ, Moore JP.2015. Influences on the design and purification of soluble, recombinant native-like HIV-1 envelope glycoprotein trimers. J Virol 89:12189 -12210. doi:10.1128 / JVI.01768-15.

[0231] In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signalAttorney Docket: 2933311.091.WO1 DU8448PCT peptide of SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the recombinant envelope trimer includes three copies of a recombinant HIV-1 envelope including all the consecutive amino acids after the signal peptide of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24. Multimeric envelopes

[0232] Presentation of antigens as particulates reduces the B cell receptor affinity necessary for signal transduction and expansion (See Baptista et al. EMBO J.2000 Feb 15; 19(4): 513–520). Displaying multiple copies of the antigen on a particle provides an avidity effect that can overcome the low affinity between the antigen and B cell receptor. The initial B cell receptor specific for pathogens can be low affinity, which precludes vaccines from being able to stimulate and expand B cells of interest. In particular, very few naïve B cells from which HIV-1 broadly neutralizing antibodies arise can bind to soluble HIV-1 Envelope. Provided are envelopes, including but not limited to trimers as particulate, high-density array on liposomes or other particles, for example but not limited to nanoparticles. See e.g., He et al. Nature Communications 7, Article number: 12041 (2016), doi:10.1038 / ncomms12041; Bamrungsap et al. Nanomedicine, 2012, 7 (8), 1253-1271.

[0233] To improve the interaction between the naïve B cell receptor and immunogens, envelope designs can be created wherein the envelope is presented on particles, e.g., but not limited to nanoparticle. In some embodiments, the HIV-1 Envelope trimer could be fused to ferritin. Ferritin protein self assembles into a small nanoparticle with three-fold axis of symmetry. At these axes the envelope protein is fused. Therefore, the assembly of the three- fold axis also clusters three HIV-1 envelope protomers together to form an envelope trimer. Each ferritin particle has 8 axes which equates to 8 trimers being displayed per particle. See e.g., Sliepen et al. Retrovirology201512:82, DOI: 10.1186 / s12977-015-0210-4.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0234] Any suitable ferritin sequence could be used. In non-limiting embodiments, ferritin sequences are disclosed in US Patent 10,961,283, incorporated herein by reference.

[0235] Ferritin nanoparticle linkers: The ability to form HIV-1 envelope ferritin nanoparticles relies on self-assembly of 24 ferritin subunits into a single ferritin nanoparticle. The addition of a ferritin subunit to the C-terminus of HIV-1 envelope may interfere with the ability of the ferritin subunit to fold properly and or associate with other ferritin subunits. When expressed alone, ferritin readily forms 24-subunit nanoparticles, however appending it to envelope only yields nanoparticles for certain envelopes. Since the ferritin nanoparticle forms in the absence of envelope, the envelope could be sterically hindering the association of ferritin subunits. Thus, we designed ferritin with elongated glycine-serine linkers to further distance the envelope from the ferritin subunit. To make sure that the glycine linker is attached to ferritin at the correct position, we created constructs that attach at second amino acid position or the fifth amino acid position. The first four N-terminal amino acids of natural Helicobacter pylori ferritin are not needed for nanoparticle formation but may be important for proper folding and oligomerization when appended to envelope. Thus, we designed constructs with and without the leucine, serine, and lysine amino acids following the glycine- serine linker. The goal will be to find a linker length that is suitable for formation of envelope nanoparticles when ferritin is appended to most envelopes. Any suitable linker between the envelope and ferritin could be used, so long as the fusion protein is expressed and the trimer is formed.

[0236] Another approach to multimerize expression constructs uses Staphylococcus Sortase A transpeptidase ligation to conjugate inventive envelope trimers, for e.g. but not limited to cholesterol. The trimers can then be embedded into liposomes via the conjugated cholesterol. To conjugate the trimer to cholesterol either a C-terminal LPXTG tag (SEQ ID NO: 15) or a N-terminal pentaglycine repeat tag GGGGG (SEQ ID NO: 16) is added to the envelope trimer gene. Cholesterol is also synthesized with these two tags. Sortase A is then used to covalently bond the tagged envelope to the cholesterol. The sortase A-tagged trimer protein can also be used to conjugate the trimer to other peptides, proteins, or fluorescent labels. In non-limiting embodiments, the sortase A tagged trimers are conjugated to ferritin to form nanoparticles. Any suitable ferritin can be used.

[0237] The invention provides design of envelopes and trimer designs wherein the envelope comprises a linker which permits addition of a lipid, such as but not limited toAttorney Docket: 2933311.091.WO1 DU8448PCT cholesterol, via a Sortase A reaction. See e.g. Tsukiji, S. and Nagamune, T. (2009), Sortase- Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering. ChemBioChem, 10: 787–798. Doi:10.1002 / cbic.200800724; Proft, T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification andimmobilization. Biotechnol Lett (2010) 32: 1. Doi:10.1007 / s10529-009-0116-0; Lena Schmohl, Dirk Schwarzer, Sortase-mediated ligations for the site-specific modification of proteins, Current Opinion in Chemical Biology, Volume 22, October 2014, Pages 122-128, ISSN 1367-5931, dx.doi.org / 10.1016 / j.cbpa.2014.09.020; Tabata et al. Anticancer Res.2015 Aug;35(8):4411- 7; Pritz et al. J. Org. Chem.2007, 72, 3909-3912.

[0238] The lipid modified envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0239] The lipid modified and multimerized envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0240] The trimer could be incorporated in a nanoparticle, including without limitation any ferritin-based nanoparticle.

[0241] Throughout the application amino acid positions numbers refer to HXB2 numbering.

[0242] Any of the immunogens herein may be encoded by a nucleic acid. It will be understood that non-identical nucleic acid sequences may encode the same amino acid sequence. As such these examples do not exclude nucleic acid sequences that encode immunogens with the same amino acid sequence but possess different nucleic acid sequences.

[0243] Modifications of nucleic acids encoding the inventive envelopes may include: ^ 5’UTR including aGcATAAAAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAG CATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTT CTGAAAATTTTCACCATTTACGAACGATAGCGCT (SEQ ID NO: 17). Without being bound by theory, this modification is an improved 5’ UTR sequence for mRNA stability and half-life from screens. See Messenger RNA-Based Vaccines Against Infectious Diseases. Alameh MG, Weissman D, Pardi N.Curr Top Microbiol Immunol.2020 Apr 17. Doi: 10.1007 / 82_2020_202. PMID: 32300916.Attorney Docket: 2933311.091.WO1 DU8448PCT ^ 3’UTR including actagtAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGA ATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCA AAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCT (SEQ ID NO: 18). Without being bound by theory, this modification is an improved 5' UTR sequence for mRNA stability and half-life from screens. See Messenger RNA-Based Vaccines Against Infectious Diseases. Alameh MG, Weissman D, Pardi N.Curr Top Microbiol Immunol.2020 Apr 17. Doi: 10.1007 / 82_2020_202. PMID: 32300916. ^ poly A (immediately after 3’UTR) includes AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 19). Without being bound by theory, this modification is an improved polyA tail sequence for mRNA stability and half-life. See Jalkanen et al. Semin Cell Dev Biol.34:24-32 (2014). ^ mRNA codon optimization includes a reverse translation of protein amino acid sequence to optimal codons. Without being bound by theory, this modification codon optimization is performed as follows: amino acid sequence is reverse translated into an DNA sequence using a modified mammalian codon usage table. The table increases both the CIA and the GC content of the mRNA. The reverse translated sequence (or mRNA sequence) is modeled into mFold and Delta H / Delta G computed, and the sequence with the lowest free energy is selected. In some cases, the codons can be replaced in specific locations to relax the tridimentional structure of the optimized mRNA. The sequence is then cloned between the 5’UTR and 3’UTR above. See Leppek et al. Nature Communications 13:1536 (2022). In addition, expression of any of the mRNAs of the present invention described herein can be enhanced by synthesis of a branched polyA tail as described in Chen, et al., Nat. Biotech. doi.org / 10.1038 / s41587-024-02174-7 (2024).

[0244] The exemplary constructs provided herein, include various combinations of these modifications. Any modification or combination of the modifications described herein, including but not limited, to different versions of soluble proteins, different versions of membrane expressed proteins, stabilization mutations, furin cleavage site mutations, signal peptides, and / or cytoplasmic tail modifications can be applied to any HIV-1 envelope protein sequence described herein.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0245] In non-limiting embodiments, sortase A tagged recombinant envelope trimers (e.g., any of the recombinant envelopes described in Table 1) can be displayed on the surface of sortase A conjugated ferritin nanoparticles.

[0246] Described herein are multimers including one or more of any of the recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers in a nanoparticle) included in Table 1. In some embodiments, the multimer includes one or more of any of the recombinant envelope trimers including the amino acid sequence set forth in any one of SEQ ID NO: 1-5 and 21-24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences SEQ ID NO: 1- 5 and 21-24. In some embodiments, the multimer is a nanoparticle including one or more of any of the recombinant envelope trimers including the amino acid sequence set forth in any one of SEQ ID NO: 1-5 and 21-24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences SEQ ID NO: 1-5 and 21-24, or a combination thereof.

[0247] In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ IDAttorney Docket: 2933311.091.WO1 DU8448PCT NO: 5. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0248] In some embodiments, the multimer is a ferritin nanoparticle that includes one or more of any of the recombinant envelope trimers including the amino acid sequence set forth in any one of SEQ ID NO: 1-5 and 21-24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences SEQ ID NO: 1-5 and 21-24, or a combination thereof.

[0249] In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence setAttorney Docket: 2933311.091.WO1 DU8448PCT forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle. In some embodiments, a recombinant envelope trimer including the amino acidAttorney Docket: 2933311.091.WO1 DU8448PCT sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 can be displayed as a multimer including one or more recombinant envelope trimers (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 trimers) in a nanoparticle, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

[0250] In some embodiments, any of the envelope trimers described herein can be lipid modified as described herein. In some embodiment, any of the amino acid sequences including (SEQ ID NOs: 1-10, 21-25) can be lipid modified as described herein. In some embodiments, the multimer is composed in a lipid nanoparticle including one or more of any of the recombinant envelope trimers including the amino acid sequence set forth in any one of SEQ ID NO: 1-5, and 21-24 or the nucleic acid set forth in any one of SEQ ID NO: 6-10 and 25, or an amino acid or nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences SEQ ID NO: 1-10 and 21-24, or a combination thereof. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 2 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%Attorney Docket: 2933311.091.WO1 DU8448PCT sequence identity to the nucleotide sequence of SEQ ID NO: 5 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 21 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 22 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 23 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide sequence encoding for the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 24 can be displayed and / or encapsulated in a lipid nanoparticle.

[0251] In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 6 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 7 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 8 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 9 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 9 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments,Attorney Docket: 2933311.091.WO1 DU8448PCT mRNA encoded by the nucleotide set forth in SEQ ID NO: 10 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 10 can be displayed and / or encapsulated in a lipid nanoparticle. In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 25 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 25 can be displayed and / or encapsulated in a lipid nanoparticle. Compositions

[0252] In certain aspects, the invention provides compositions of CH505 Envs, as gp120s, gp140s cleaved and uncleaved, gp145s, gp150s and gp160s, stabilized and / or multimerized trimers, as proteins, DNAs, RNAs, or any combination thereof, administered as primes and boosts to elicit immune response. CH505 Envs as proteins would be co- administered with nucleic acid vectors containing Envs to amplify antibody induction. In certain embodiments, the compositions and methods include any immunogenic HIV-1 sequences to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic and / or consensus HIV-1 genes to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic group M and / or consensus genes to give the best coverage for T cell help and cytotoxic T cell induction. In some embodiments, the mosaic genes are any suitable gene from the HIV-1 genome. In some embodiments, the mosaic genes are Env genes, Gag genes, Pol genes, Nef genes, or any combination thereof. See, e.g. U.S. Patent No.7951377. In some embodiments the mosaic genes are bivalent mosaics. In some embodiments the mosaic genes are trivalent. In some embodiments, the mosaic genes are administered in a suitable vector with each immunization with Env gene inserts in a suitable vector and / or as a protein. In some embodiments, the mosaic genes, for example as bivalent mosaic Gag group M consensus genes, are administered in a suitable vector, for example but not limited to HSV2, would be administered with each immunization with Env gene inserts in a suitable vector, for example but not limited to HSV-2.

[0253] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of any one of SEQ ID NOs: 1-5 and 21-24 or anAttorney Docket: 2933311.091.WO1 DU8448PCT amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NOs: 1-5 and 21-24; and optionally, a carrier.

[0254] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; and optionally, a carrier.

[0255] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2; and optionally, a carrier.

[0256] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3; and optionally, a carrier.

[0257] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4; and optionally, a carrier.

[0258] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5; and optionally, a carrier.

[0259] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21; and optionally, a carrier.

[0260] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; and optionally, a carrier.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0261] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23; and optionally, a carrier.

[0262] In some embodiments, the composition includes an HIV envelope including all the amino acids after the signal peptide of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24; and optionally, a carrier.

[0263] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in any one of SEQ ID NOs: 6-10 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6-10 and 25; and optionally, a carrier.

[0264] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 6 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6; and optionally, a carrier.

[0265] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 7 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7; and optionally, a carrier.

[0266] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 8 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8; and optionally, a carrier.

[0267] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 9 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 9; and optionally, a carrier.

[0268] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 10 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 10; and optionally, a carrier.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0269] In some embodiments, the composition includes an HIV envelope including the nucleotide sequence set forth in SEQ ID NO: 25 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 25; and optionally, a carrier.

[0270] In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acidAttorney Docket: 2933311.091.WO1 DU8448PCT sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant.

[0271] In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 6 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 7 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 8 or aAttorney Docket: 2933311.091.WO1 DU8448PCT nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 10 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant. In some embodiments, the composition includes a lipid nanoparticle including mRNA derived from the nucleic acid sequence set forth in SEQ ID NO: 25 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 25, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or and adjuvant.

[0272] In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the nanoparticleAttorney Docket: 2933311.091.WO1 DU8448PCT displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. In some embodiments, the composition includes a ferritin self-assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers. Nucleic acids

[0273] In certain aspects the invention provides a cell comprising a nucleic acid encoding any one of the envelopes of the invention suitable for recombinant expression. InAttorney Docket: 2933311.091.WO1 DU8448PCT certain aspects, the invention provides a clonally derived population of cells encoding any one of the envelopes of the invention suitable for recombinant expression. In certain aspects, the invention provides a stable pool of cells encoding any one of the envelopes of the invention suitable for recombinant expression.

[0274] In certain aspects, the invention provides a recombinant HIV-1 envelope polypeptide listed in Table 1. In certain embodiments, the polypeptide is a non-naturally occurring protomer designed to form an envelope trimer. The invention also provides nucleic acids encoding these recombinant polypeptides.

[0275] In certain aspects the invention provides a recombinant trimer comprising three identical protomers of an envelope from Table 1. In certain aspects the invention provides an immunogenic composition comprising the recombinant trimer and a carrier, wherein the trimer comprises three identical protomers of an HIV-1 envelope listed in Table 1. In certain aspects the invention provides an immunogenic composition comprising a nucleic acid encoding these recombinant HIV-1 envelope and a carrier.

[0276] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted in an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0277] In certain embodiments the induced immune response includes induction of antibodies, including but not limited to autologous and / or cross-reactive (broadly) neutralizing antibodies against HIV-1 envelope. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are known in the art and are also described herein.

[0278] In certain aspects the invention provides a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides a nucleic acid consisting essentially of a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the inventionAttorney Docket: 2933311.091.WO1 DU8448PCT provides an expression vector consisting essentially a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter.

[0279] In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector. Methods

[0280] In certain aspects the invention provides compositions and methods of Env genetic immunization either alone or with Env proteins to recreate the swarms of evolved viruses that have led to bnAb induction. Nucleotide-based vaccines offer a flexible vector format to immunize against virtually any protein antigen. Currently, two types of genetic vaccination are available for testing—DNAs and mRNAs.

[0281] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See, Graham BS, Enama ME, Nason MC, Gordon IJ, Peel SA, et al. (2013) DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for delivery of nucleic acids, as DNA and / or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments, DNA is formulated for delivery by a gene gun. In certain embodiments, DNA is administered by electroporation, or by a needle-free injection technology, for example but not limited to Biojector® device. In certain embodiments, the DNA is inserted in vectors. The DNA is delivered using a suitable vector for expression in mammalian cells. In certain embodiments the nucleic acids encoding the envelopes are optimized for expression. In certain embodiments DNA is optimized, e.g. codon optimized, for expression. In certainAttorney Docket: 2933311.091.WO1 DU8448PCT embodiments the nucleic acids are optimized for expression in vectors and / or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus-based vectors, rAdenovirus (e.g. Barouch DH, et al. Nature Med.16: 319-23, 2010), recombinant mycobacteria (e.g. rBCG or M smegmatis) (Yu, JS et al. Clinical Vaccine Immunol.14: 886- 093,2007; ibid 13: 1204-11,2006), and recombinant vaccinia type of vectors (Santra S. Nature Med.16: 324-8, 2010), for example but not limited to ALVAC, replicating (Kibler KV et al., PLoS One 6: e25674, 2011 nov 9.) and non-replicating (Perreau M et al. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MVA)), adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons, Herpes Simplex Virus vectors, and other suitable vectors.

[0282] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies which contemplate using DNA or RNA or may use complexes of nucleic acid molecules and other entities to be used in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer (amphiphilic block copolymer 704). See Cany et al., Journal of Hepatology 2011 vol.54 j 115–121; Arnaoty et al., Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology, vol.859, pp293-305 (2012); Arnaoty et al. (2013) Mol Genet Genomics.2013 Aug;288(7-8):347-63. Nanocarrier technologies called Nanotaxi® for immunogenic macromolecules (DNA, RNA, Protein) delivery are under development. See for example technologies developed by incellart.

[0283] In certain aspects, the invention provides nucleic acids comprising sequences encoding envelopes of the invention. In certain embodiments, the nucleic acids are DNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0284] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0285] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising aAttorney Docket: 2933311.091.WO1 DU8448PCT nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector.

[0286] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0287] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the inventive sequences. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of the sequences of the invention, or a variant thereof or a fragment thereof. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of inventive antibodies. The RNA may be plus- stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.

[0288] In some embodiments, a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp, CleanCap® (e.g., the AG, GG, AU, 3’OMe AG, or 3’OMe GG CleanCap®), or ARCA). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7- methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. InAttorney Docket: 2933311.091.WO1 DU8448PCT some embodiments, a RNA molecule useful with the invention may be single-stranded. In some embodiments, a RNA molecule useful with the invention may comprise synthetic RNA.

[0289] The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the envelope. Optimization can also improve transcription and / or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a Kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

[0290] Methods for in vitro transfection of mRNA and detection of envelope expression are known in the art.

[0291] Methods for expression and immunogenicity determination of nucleic acid encoded envelopes are known in the art.

[0292] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins, including trimers such as but not limited to SOSIP based trimers, suitable for use in immunization are known in the art. In certain embodiments recombinant proteins are produced in CHO cells.

[0293] The immunogenic envelopes can also be administered as a protein prime in combination with a variety of nucleic acid envelope boosts (e.g., HIV -1 Envs delivered as DNA expressed in viral or bacterial vectors).

[0294] Dosing of proteins and nucleic acids can be readily determined by a skilled artisan. A single dose of nucleic acid can range from a few nanograms (ng) to a few micrograms (μg) or milligram of a single immunogenic nucleic acid. Recombinant protein dose can range from a few μg micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide.

[0295] Administration: The compositions can be formulated in designs that incorporate appropriate carriers such as peptides for enhancing CD4+ T cell help, known as PADRE, GTH1, GTH2, or any combination thereof. In certain embodiments the compositions are delivered via intramuscular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes, or any other suitable route of immunization.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0296] The compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to, alum, 3M052, poly IC, MF-59 or other squalene-based adjuvant, AS01B, or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen (Leroux- Roels et al., IABS Conference, April 2013). In certain embodiments, TLR agonists are used as adjuvants. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions.

[0297] In certain embodiments, the compositions and methods comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In non-limiting embodiments modulation includes PD- 1 blockade; T regulatory cell depletion; CD40L hyperstimulation; soluble antigen administration, wherein the soluble antigen is designed such that the soluble agent eliminates B cells targeting dominant epitopes, or a combination thereof. In certain embodiments, an immunomodulatory agent is administered in at time and in an amount sufficient for transient modulation of the subject's immune response so as to induce an immune response which comprises broad neutralizing antibodies against HIV-1 envelope. Non-limiting examples of such agents is any one of the agents described herein: e.g. chloroquine (CQ), PTP1B Inhibitor - CAS 765317-72-4 - Calbiochem or MSI 1436 clodronate or any other bisphosphonate; a Foxo1 inhibitor, e.g.344355 | Foxo1 Inhibitor, AS1842856 - Calbiochem; Gleevac, anti- CD25 antibody, anti-CCR4 Ab, an agent which binds to a B cell receptor for a dominant HIV-1 envelope epitope, or any combination thereof. In non-limiting embodiments, the modulation includes administering an anti-CTLA4 antibody. Non-limiting examples are ipilimumab and tremelimumab. In certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different.

[0298] There are various host mechanisms that control bnAbs. For example, highly somatically mutated antibodies become autoreactive and / or less fit (Immunity 8: 751, 1998; PloS Comp. Biol.6 e1000800, 2010; J. Thoret. Biol.164:37, 1993); Polyreactive / autoreactive naïve B cell receptors (unmutated common ancestors of clonal lineages) can lead to deletionAttorney Docket: 2933311.091.WO1 DU8448PCT of Ab precursors (Nature 373: 252, 1995; PNAS 107: 181, 2010; J. Immunol.187: 3785, 2011); Abs with long HCDR3 can be limited by tolerance deletion (JI 162: 6060, 1999; JCI 108: 879, 2001). BnAb knock-in mouse models are providing insights into the various mechanisms of tolerance control of MPER BnAb induction (deletion, anergy, receptor editing). Other variations of tolerance control likely will be operative in limiting BnAbs with long HCDR3s, high levels of somatic hypermutations.

[0299] For a summary of CH505 sequences and designs see US Patent 10,968,255, for example, but not limited to, Table 1, Figures 22-24, and US Patent 10,004,800 (Figure 17), the contents of each of which are hereby incorporated by reference in their entireties.

[0300] It is readily understood that the envelope glycoproteins referenced in various examples and figures comprise a signal peptide / leader sequence. It is well known in the art that HIV-1 envelope glycoprotein is a secretory protein with a signal peptide or leader sequence that is removed during processing and recombinant expression (without removal of the signal peptide, the protein is not secreted). See for example Li et al. Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences. Virology 204(1):266-78 (1994) (“Li et al.1994”), at first paragraph, and Li et al. Effects of inefficient cleavage of the signal sequence of HIV-1 gpl20 on its association with calnexin, folding, and intracellular transport. PNAS 93:9606-9611 (1996) (“Li et al. 1996”), at 9609. Any suitable signal peptide sequence could be used. In some embodiments the leader sequence is the endogenous leader sequence. Most of the gp120 and gp160 amino acid sequences include the endogenous leader sequence. In other non-limiting examples, the leader sequence is human Tissue Plasminogen Activator (TPA) sequence, human CD5 leader sequence (e.g. MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO: 20)). Most of the chimeric designs include CD5 leader sequence. A skilled artisan appreciates that when used as immunogens, and for example when recombinantly produced, the amino acid sequences of these proteins do not comprise the signal peptide / leader sequences.

[0301] In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the methods of inducing an immune response in a subject includeAttorney Docket: 2933311.091.WO1 DU8448PCT administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, theAttorney Docket: 2933311.091.WO1 DU8448PCT methods of inducing an immune response in a subject include administering nanoparticles including the amino acid sequence or mRNA derived from the nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0302] In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 6 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 7 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 8 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 10 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the methods of inducing an immune response in a subject include administering nanoparticles including mRNA encoded by the nucleic acid sequence set forth in SEQ ID NO: 25 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 25.

[0303] In some embodiments, the methods of inducing an immune response in a subject include any of the compositions described herein in an amount sufficient to induce anAttorney Docket: 2933311.091.WO1 DU8448PCT immune response. In some embodiments, the methods of inducing an immune response in a subject include administering an immunogenic composition comprising any one of the recombinant envelopes described herein (e.g., Table 1) and / or any of the compositions described herein in an amount sufficient to induce an immune response.

[0304] In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequenceAttorney Docket: 2933311.091.WO1 DU8448PCT having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 5 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 23 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the amino acid sequence, or the mRNA encoded by the nucleic acid encoding for the amino acid sequence set forth in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24 in an amount sufficient to induce an immune response in a subject.

[0305] In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%Attorney Docket: 2933311.091.WO1 DU8448PCT sequence identity to the amino acid sequence of SEQ ID NO: 6 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 7 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 8 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 9 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 in an amount sufficient to induce an immune response in a subject. In some embodiments, the methods of inducing an immune response in a subject include administering a composition including a recombinant envelope and / or a nanoparticle including the nucleic acid sequence, or the mRNA derived from the nucleic acid corresponding to the nucleic acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,Attorney Docket: 2933311.091.WO1 DU8448PCT 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25 in an amount sufficient to induce an immune response in a subject.

[0306] In some embodiments, the methods of inducing an immune response in a subject include any of the compositions described herein in an amount sufficient to induce an immune response, wherein the composition is administered as a prime. In some embodiments, the methods of inducing an immune response in a subject include any of the compositions described herein in an amount sufficient to induce an immune response, wherein the composition is administered as a boost. In some embodiments, the methods of inducing an immune response in a subject include administering an immunogenic composition comprising any one of the recombinant envelopes described herein (e.g., Table 1) herein in an amount sufficient to induce an immune response and / or any of the compositions described herein in an amount sufficient to induce an immune response, wherein the recombinant envelope and / or the composition is administered as a boost.

[0307] In some embodiments, the methods of inducing an immune response in a subject include administering an immunogenic composition comprising any one of the recombinant envelopes described herein (e.g., Table 1) herein in an amount sufficient to induce an immune response and / or any of the compositions described herein in an amount sufficient to induce an immune response, wherein the recombinant envelope and / or the composition is administered as a prime.

[0308] In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subjectAttorney Docket: 2933311.091.WO1 DU8448PCT includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a boost.

[0309] In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as aAttorney Docket: 2933311.091.WO1 DU8448PCT boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a boost.

[0310] In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including aAttorney Docket: 2933311.091.WO1 DU8448PCT recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a boost.

[0311] In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the aminoAttorney Docket: 2933311.091.WO1 DU8448PCT acid sequence set forth in SEQ ID NO: 21 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as aAttorney Docket: 2933311.091.WO1 DU8448PCT boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 21 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a boost. In someAttorney Docket: 2933311.091.WO1 DU8448PCT embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 22 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing anAttorney Docket: 2933311.091.WO1 DU8448PCT immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 23 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 1 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 2 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 3 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 4 as a boost. In some embodiments, the method of inducing an immune response in a subject includes administering a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 24 as a prime and a composition including a nanoparticle including a recombinant envelope including the amino acid sequence set forth in SEQ ID NO: 5 as a boost.Attorney Docket: 2933311.091.WO1 DU8448PCT EXAMPLES

[0312] The Examples / Methods have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The following Examples are offered by way of illustration and not by way of limitation. EXAMPLE 1 – HIV ENVELOPES TRIMERS

[0313] Universal HIV Env priming immunogens were designed following the rationale depicted in (Figure 1). The prefusion structures of three classes of well-characterized UCA targeting Envelope immunogens are shown in Figure 1. The V1swap Env (Figure 1, left) is primarily derived from CH84810.17 but has undergone extensive modification of its V3 glycan epitope and proximal domains in order to facilitate the binding of multiple V3 glycan UCAs. Immunogens derived from the CH505 Env (Figure 1, center) have been shown to bind with high affinity to UCAs that can go on to mature into CD4 binding site (CD4bs) bnAbs. Similarly, several Env candidates have been proposed as priming immunogens to target V2 apex UCAs, including CAP256 (Figure 1, right). In an effort to combine the favorable antigenic characteristics from each of these classes of immunogens, chimeric Envelopes are under development that make use of a CH505 “backbone” onto which a variety of V3 glycan and V2 apex modifications can be grafted. EXAMPLE 2 – HIV SOLUBLE CH505pUCA AND CH505pUCA I130H CstrandOPT ECTODOMAINS

[0314] Soluble CH505pUCA and CH505pUCA I130H CstrandOPT ectodomains were generated and their expression and antigenic characterization were assessed.

[0315] Plasmids encoding for soluble CH505pUCA SOSIP-2P ectodomains and furin were co-transfected at a ratio of 4:1 into FreeStyle293 cells using polyethyleneimine. Cells were allowed to incubate for 5 days before cell supernatants were harvested and filtered.Attorney Docket: 2933311.091.WO1 DU8448PCT CH505pUCA SOSIP-2P and CH505pUCA I130H CstrandOPT SOSIP-2P were purified from filtered supernatant via affinity chromatography with PGT145 resin. Envelopes were eluted using 10mM Tris pH 8.0, 3M MgCl2and affinity-purified proteins were then subjected to size-exclusion chromatography using a Superose 6 Increase 10 / 300 GL column (Cytiva) in 2mM Tris pH 8.0, 200mM NaCl, 0.02% NaN3.

[0316] The expression of soluble CH505pUCA and CH505pUCA I130H CstrandOPT ectodomains were assessed by size-exclusion chromatography. Figure 2A shows a chromatogram of the CH505pUCA Env after PGT145 affinity chromatography. The profile from this Superose 6 Increase 10-300 column shows a homogeneous peak at the expected elution volume for a densely glycosylated prefusion trimer. Figure 5A shows a chromatogram of the CH505pUCA I130H CstrandOPT Env after PGT145 affinity chromatography. The profile from this Superose6 Increase 10-300 column shows a homogeneous peak at the expected elution volume for a densely glycosylated prefusion trimer

[0317] The antigenicity of CH505pUCA and CH505pUCA I130H CstrandOPT Env were assessed by biolayer interferometry. CH505pUCA SOSIP-2P and CH505pUCA I130H CstrandOPT constructs with a C-terminal 8xHisTag were immobilized to NiNTA sensortips (Sartorius) at a response rate of 1.6nm. Sensortips were then dipped into wells containing monoclonal antibodies at concentrations of 100nM. Data were collected using an Octet RED96e System (Sartorius). Data were reference subtracted and analyzed using Octet Data Analysis Software v12.0.

[0318] As shown in Figure 2B, although the CH505pUCA Env had not yet been modified to enhance targeting of V2 apex UCAs, it still exhibits detectable binding to the reverted unmutated ancestor (RUA) of the V2-directed bnAb CH01. Further modification to enhance reactivity against the V2 apex epitope (I130H / I161A / E170R / K172E / N173Y) results in the CH505pUCA I130H CstrandOPT construct, which now shows additional reactivity against the PG16 RUA V2 bnAb precursor without losing binding to the CH01 RUA, as shown in Figure 5B. EXAMPLE 3 – HIV TRANSMEMBRANE-IMMOBILIZED CH505pUCA CONSTRUCTS

[0319] Transmembrane-immobilized CH505pUCA constructs were generated and their expression levels assessed by flow cytometry.Attorney Docket: 2933311.091.WO1 DU8448PCT

[0320] Plasmids encoding for CH505pUCA gp150712, CH505pUCA gp150755, CH505pUCA gp150 TM1 or CH505pUCA gp160 were transfected into FreeStyle293 cells using jetPRIME (Polyplus). Transfected cells were allowed to express for 2 days before being pelleted at 500g and wash with 1% BSA in PBS. Cells were then resuspended to a density of 1.0 x106in 1% BSA (PBS).50,000 cells were aliquoted to each well of U-bottom 96-well plates. An equal volume of monoclonal antibodies was added to cells for a final concentration of 2 μg / mL. These antibodies were incubated with cells at 4°C for 30 min to allow binding to occur. Cells were then washed once with 1% BSA (PBS) before being incubated with goat anti-human IgG Fc-PE (Thermo Fisher) at a final concentration of 2.5 μg / mL in 1% BSA / PBS. Cells were the incubated for 30 minutes at 4°C while protected from light. Cells were washed once with PBS before being incubated with LIVE / DEAD fixable aqua dead cell stain (Thermo Fisher) for 20 minutes at room temperature. Cells were once again protected from light during this incubation. Finally, cells were washed once with 1% BSA (PBS) and resuspended in 1% BSA, 2 mM EDTA, and 1% paraformaldehyde in PBS. Flow cytometric data were acquired and analyzed on an iQue 3 high-throughput flow cytometry system (Sartorius).

[0321] As shown in Figure 3, mean fluorescence intensities (MFI) plotted for each cell population after being stained with the mAb listed above each plot indicates that CH505pUCA gp150712, CH505pUCA gp150755, CH505pUCA gp150 TM1 or CH505pUCA gp160 are expressed in FreeStyle293 cells.

[0322] The antigenicity of transmembrane CH505pUCA Envelope was assessed. As shown in Figure 4, cells transfected with the CH505pUCA gp150755 construct (DW-198) were evaluated for binding to a panel of the 24 mAbs listed on the x-axis. Antibodies are grouped based on their respective binding characteristics in 5 groups: CD4 binding site UCAs, V3 glycan UCAs, V2 apex UCAs, broadly neutralizing antibodies (bnAbs), and base- binders and non-neutralizing antibodies (nonAbs). The influenza HA-directed mAb CH65 is colored black and was included as a negative control. The y-axis plots MFI after staining with each mAb. The influenza HA-directed mAb CH65 was included as a negative control.

[0323] The data in Figure 4 show that the transmembrane, cell surface-expressed CH505pUCA Envelope exhibits substantially the same antigenicity as the soluble ectodomain that was evaluated and the data shown in Figure 2. In Figure 2, the data showed that the modifications were effective in generating an immunogen capable of binding to UCAs thatAttorney Docket: 2933311.091.WO1 DU8448PCT target multiple antigenic sites (e.g., CD4 binding site, V3 glycan and V2 apex). In Figure 4, the data show that the favorable antigenic characteristics are retained on the cell surface.

[0324] The antigenicity of transmembrane CH505pUCA I130H CstrandOPT Envelopes was assessed. As shown in Figure 6, cells transfected with the CH505pUCA I130H CstrandOPT_gp150_755 construct (HV1304323) and CH505pUCA I130H CstrandOPT_eFP_MPER656_gp150_755 construct (HV1304324) were evaluated against MPER.B-TMD Y712I (HV1303006) and V1swap (HV1303483) antigens for binding to a panel of the 24 mAbs listed on each graph. The antibodies tested were: CH235 UCAtkLL is a CD4 binding site bnAb precursor; BG18 UCA, DH270 UCA3, BF520.1 UCA and PCDN76 UCA are V3 glycan bnAb precursors; DH511 UCA, DH511delta96_UCA, 2F5_RUA-D and 2F5_RUA-N are MPER bnAb precursors; 2G12 and PGT128 are mature V3 glycan bnAbs; N6 and CH235.12 are mature CD4 binding site bnAbs; PGT145 is a mature V2 apex bnAb; PGT151 is a mature fusion domain bnAb; DH511, 2F5 IgG1, Ab 10E8 and Ab 4E10 are mature MPER bnAbs; RM19R IgG is a base-binding antibody; and A32, 17b and 19b are non-neutralizing antibodies. The influenza HA-directed mAb CH65 is colored black and was included as a negative control. The y-axis plots MFI after staining with each mAb. The influenza HA-directed mAb CH65 was included as a negative control. HV1303006 serves as a positive control for MPER bnAbs and HV1303483 serves as a positive control for BG18 UCA, DH270 UCA3 and BF520.1 UCA binding.

[0325] As demonstrated in Figure 5B, the V2 optimization that generated the CH505pUCA I130H CstrandOPT construct results in detectable binding to PG16 RUA (Duke), a V2 apex bnAb precursor. In Figure 6, cells transfected with mRNAs encoding for transmembrane CH505pUCA I130H CstrandOPT Envs show that this V2 optimization does not diminish the ability of this Env to engage bnAb precursors that target the CD4 binding site or the V3 glycan epitope. Diminished DH270 UCA3 reactivity for these constructs relative to the CH505pUCA gp150755 construct evaluated in Figure 4 can likely be attributed to decreases in Env cell surface expression after the introduction of the V2 optimization substitutions. SEQUENCES

[0326] DW-197: CH505pUCA_gp150_712 (SEQ ID NO: 1)Attorney Docket: 2933311.091.WO1 DU8448PCT

[0327] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLICTNYAPKLRGMMRGEIKNCSFNITTELRDKREK KNALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCNN KTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLNE SVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGI

[0328] DW-198: CH505pUCA_gp150_755 (SEQ ID NO: 2)

[0329] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLICTNYAPKLRGMMRGEIKNCSFNITTELRDKREK KNALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCNN KTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLNE SVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGISPLSLQTLTPNPREPDRLRGIEEEGGEQDRDRSIRLVSGF LPI

[0330] DW-199: CH505pUCA_gp150_TM1 (SEQ ID NO: 3)Attorney Docket: 2933311.091.WO1 DU8448PCT

[0331] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLICTNYAPKLRGMMRGEIKNCSFNITTELRDKREK KNALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCNN KTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLNE SVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGIRPVFSSPPSYF

[0332] DW-200: CH505pUCA_gp160 (SEQ ID NO: 4)

[0333] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLICTNYAPKLRGMMRGEIKNCSFNITTELRDKREK KNALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCNN KTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLNE SVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGISPLSLQTLTPNPREPDRLRGIEEEGGEQDRDRSIRLVSGF LPIVWDDLRSLCLFSYHRLRDFLLLAARVVELLGRSSLRGLQRGWEVLKYLGSLVQY WGLELKKSAISLFDTLAIAVAEGTDRIIELIQGFCRAIRNIPTRIRQGFEASAA

[0334] DW-205: CH505pUCA_SOSIP-2P (SEQ ID NO: 5)Attorney Docket: 2933311.091.WO1 DU8448PCT

[0335] MGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKEAKTTLFC ASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMHED VISLWDQSLKPCVKLTPLCVTLICTNYAPKLRGMMRGEIKNCSFNITTELRDKREKKN ALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCNNKT FTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLNESV KIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKLKE YFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTETNS TRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRPG GGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFLG AAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQARV LAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEIS NYTQIIYGLLEESQNQQEKNEQDLLALD

[0336] DW-197: CH505pUCA_gp150_712 (DNA) (SEQ ID NO: 6)

[0337] ATGCGGCCTACTTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGCTAGC TCTGTGGGCACCCGCTAGAGGGGCTGAAAATCTCTGGGTCACCGTTTATTACGGT GTACCAGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAA GCTTATGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACC GATCCCAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGT GGAAAAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACC AATCCCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCATATGTAC GAACTATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTC CTTTAATATCACGACTGAACTTAGGGACAAGCGAGAAAAGAAGAACGCTCTCTT TTATAAGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATC AATTGCGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCA TTCCAATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAA GACCTTTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACAC GGAATAAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAG GGTGAGATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATC GTTCACCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACG AGAACCCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATA ATCGGCGACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAGAttorney Docket: 2933311.091.WO1 DU8448PCT ACCCTCCAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTA CGTTTCAACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTG TGGCGGAGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATG GCTAATAGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCA CCATTCAGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAG CAATGTATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGG CCTGCTGTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGG AGGTGGAAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGT AAAGATTGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGG CCGGAGAAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCT GGGAGCCGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGC ACGCAATCTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCA GAACCGCAGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAA GCAAGGGTACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTT GGGGTTGTAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATG GTCCAACCGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGAC AAAGAGATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAG AACCAGCAGGAAAAAAATGAGCAAGATCTTCTGGCATTGGATTCATGGAACTCA CTTTGGAACTGGTTTTCTATTACAAAATGGCTGTGGTATATAAAAATCTTTATCAT GATCGTTGGGGGGCTGATAGGTCTTAGGATTGTCTTTGCCGTTTTGTCCATTGTCA ACCGGGTAAGGCAAGGTATC

[0338] DW-198: CH505pUCA_gp150_755 (DNA) (SEQ ID NO: 7)

[0339] ATGCGGCCTACTTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGCTAGC TCTGTGGGCACCCGCTAGAGGGGCTGAAAATCTCTGGGTCACCGTTTATTACGGT GTACCAGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAA GCTTATGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACC GATCCCAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGT GGAAAAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACC AATCCCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCATATGTAC GAACTATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTC CTTTAATATCACGACTGAACTTAGGGACAAGCGAGAAAAGAAGAACGCTCTCTTAttorney Docket: 2933311.091.WO1 DU8448PCT TTATAAGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATC AATTGCGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCA TTCCAATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAA GACCTTTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACAC GGAATAAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAG GGTGAGATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATC GTTCACCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACG AGAACCCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATA ATCGGCGACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAG ACCCTCCAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTA CGTTTCAACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTG TGGCGGAGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATG GCTAATAGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCA CCATTCAGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAG CAATGTATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGG CCTGCTGTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGG AGGTGGAAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGT AAAGATTGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGG CCGGAGAAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCT GGGAGCCGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGC ACGCAATCTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCA GAACCGCAGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAA GCAAGGGTACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTT GGGGTTGTAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATG GTCCAACCGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGAC AAAGAGATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAG AACCAGCAGGAAAAAAATGAGCAAGATCTTCTGGCATTGGATAGTTGGAATTCT CTTTGGAATTGGTTTAGCATCACGAAGTGGCTGTGGTACATCAAAATTTTTATCA TGATAGTGGGCGGACTCATAGGACTTCGAATCGTGTTTGCCGTCCTGAGTATCGT AAATCGCGTACGGCAAGGTATTTCACCTTTGAGCTTGCAGACCCTTACTCCCAAC CCGCGGGAGCCAGACAGACTCCGGGGTATAGAGGAAGAGGGAGGAGAACAGGA CAGGGACCGGTCCATACGCCTCGTTAGTGGATTTCTTCCGATCAttorney Docket: 2933311.091.WO1 DU8448PCT

[0340] DW-199: CH505pUCA_gp150_TM1 (DNA) (SEQ ID NO: 8)

[0341] ATGCGGCCTACTTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGCTAGC TCTGTGGGCACCCGCTAGAGGGGCTGAAAATCTCTGGGTCACCGTTTATTACGGT GTACCAGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAA GCTTATGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACC GATCCCAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGT GGAAAAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACC AATCCCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCATATGTAC GAACTATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTC CTTTAATATCACGACTGAACTTAGGGACAAGCGAGAAAAGAAGAACGCTCTCTT TTATAAGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATC AATTGCGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCA TTCCAATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAA GACCTTTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACAC GGAATAAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAG GGTGAGATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATC GTTCACCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACG AGAACCCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATA ATCGGCGACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAG ACCCTCCAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTA CGTTTCAACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTG TGGCGGAGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATG GCTAATAGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCA CCATTCAGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAG CAATGTATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGG CCTGCTGTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGG AGGTGGAAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGT AAAGATTGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGG CCGGAGAAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCT GGGAGCCGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGC ACGCAATCTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCAAttorney Docket: 2933311.091.WO1 DU8448PCT GAACCGCAGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAA GCAAGGGTACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTT GGGGTTGTAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATG GTCCAACCGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGAC AAAGAGATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAG AACCAGCAGGAAAAAAATGAGCAAGATCTTCTGGCATTGGATTCTTGGAATAGT CTTTGGAATTGGTTCAGCATCACTAAATGGCTTTGGTACATAAAAATATTTATTAT GATAGTCGGAGGGCTTATTGGTTTGCGGATAGTATTTGCGGTCTTGTCTATCGTTA ACCGGGTCAGACAAGGAATTCGCCCGGTTTTTTCATCCCCCCCGAGCTATTTT

[0342] DW-200: CH505pUCA_gp160 (DNA) (SEQ ID NO: 9)

[0343] ATGCGGCCTACTTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGCTAGC TCTGTGGGCACCCGCTAGAGGGGCTGAAAATCTCTGGGTCACCGTTTATTACGGT GTACCAGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAA GCTTATGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACC GATCCCAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGT GGAAAAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACC AATCCCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCATATGTAC GAACTATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTC CTTTAATATCACGACTGAACTTAGGGACAAGCGAGAAAAGAAGAACGCTCTCTT TTATAAGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATC AATTGCGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCA TTCCAATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAA GACCTTTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACAC GGAATAAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAG GGTGAGATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATC GTTCACCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACG AGAACCCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATA ATCGGCGACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAG ACCCTCCAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTA CGTTTCAACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTG TGGCGGAGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATGAttorney Docket: 2933311.091.WO1 DU8448PCT GCTAATAGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCA CCATTCAGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAG CAATGTATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGG CCTGCTGTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGG AGGTGGAAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGT AAAGATTGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGG CCGGAGAAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCT GGGAGCCGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGC ACGCAATCTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCA GAACCGCAGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAA GCAAGGGTACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTT GGGGTTGTAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATG GTCCAACCGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGAC AAAGAGATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAG AACCAGCAGGAAAAAAATGAGCAAGATCTTCTGGCATTGGATTCATGGAATTCT TTGTGGAATTGGTTTAGTATAACAAAATGGCTGTGGTACATCAAAATCTTTATAA TGATAGTTGGAGGATTGATAGGCCTCCGAATTGTCTTTGCGGTTTTGAGCATTGT GAACCGCGTGAGGCAAGGCATATCTCCCCTCTCCTTGCAAACTTTGACGCCGAAT CCTCGCGAGCCCGATAGGCTTCGAGGAATTGAGGAGGAGGGTGGGGAGCAAGAC CGCGACCGCTCCATTAGATTGGTAAGCGGCTTTTTGCCGATAGTTTGGGATGATC TGCGATCACTTTGCCTTTTTTCTTACCACCGACTCAGAGATTTCCTCCTCTTGGCC GCCAGGGTAGTCGAGCTCTTGGGTCGCTCTTCCCTCCGCGGCCTTCAACGCGGGT GGGAAGTTCTCAAATATTTGGGTTCACTGGTCCAATATTGGGGCTTGGAATTGAA GAAGTCAGCAATAAGCCTGTTCGATACTCTCGCTATCGCGGTCGCAGAGGGGACT GATCGGATAATAGAGTTGATTCAAGGTTTTTGTAGGGCCATCAGGAACATCCCGA CAAGGATTAGGCAAGGCTTCGAAGCCAGTGCAGCT

[0344] DW-205: CH505pUCA_SOSIP-2P (DNA) (SEQ ID NO: 10)

[0345] ATGGGTAGTCTTCAACCCCTTGCTACCCTTTACCTTCTCGGTATGTTG GTTGCTTCAGTACTGGCTGCTGAAAATCTCTGGGTCACCGTTTATTACGGTGTACC AGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAAGCTTA TGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACCGATCCAttorney Docket: 2933311.091.WO1 DU8448PCT CAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGTGGAA AAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACCAATC CCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCATATGTACGAAC TATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTCCTTTA ATATCACGACTGAACTTAGGGACAAGCGAGAAAAGAAGAACGCTCTCTTTTATA AGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATCAATTG CGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCATTCCA ATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAAGACCT TTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACACGGAAT AAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAGGGTGA GATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATCGTTCA CCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACGAGAAC CCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATAATCGGC GACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAGACCCTC CAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTACGTTTC AACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTGTGGCGG AGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATGGCTAAT AGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCACCATTC AGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAGCAATGT ATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGGCCTGCT GTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGGAGGTGG AAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGTAAAGAT TGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGGCCGGAG AAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCTGGGAGC CGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGCACGCAAT CTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCAGAACCGC AGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAAGCAAGGG TACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTTGGGGTTG TAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATGGTCCAAC CGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGACAAAGAG ATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAGAACCAGC AGGAAAAAAATGAGCAAGATCTTCTGGCATTGGATAttorney Docket: 2933311.091.WO1 DU8448PCT

[0346] DW-301: CH505pUCA_I130H CstrandOPT SOSIP-2P (AA) (SEQ ID NO: 21)

[0347] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLHCTNYAPKLRGMMRGEIKNCSFNATTELRDKRR KEYALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCN NKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLN ESVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALD

[0348] DW-301: CH505pUCA_I130H CstrandOPT SOSIP-2P (DNA) (SEQ ID NO: 25)

[0349] ATGCGGCCTACTTGGGCCTGGTGGCTGTTCCTGGTGCTGCTGCTAGC TCTGTGGGCACCCGCTAGAGGGGCTGAAAATCTCTGGGTCACCGTTTATTACGGT GTACCAGTGTGGAAAGAGGCCAAAACAACGCTTTTCTGCGCATCTGACGCCAAA GCTTATGAAAAAGAGGTCCACAACGTGTGGGCAACCCACGCGTGTGTGCCAACC GATCCCAATCCCCAGGAGATGGTCCTCAAGAACGTAACAGAAAATTTTAATATGT GGAAAAACGATATGGTTGACCAGATGCACGAGGACGTTATCAGTCTCTGGGACC AATCCCTGAAGCCCTGTGTAAAACTTACACCGCTTTGCGTCACACTCCACTGTAC GAACTATGCCCCCAAGCTGAGGGGCATGATGCGGGGTGAGATCAAAAATTGCTC CTTTAATGCTACGACTGAACTTAGGGACAAGCGAAGGAAGGAATATGCTCTCTTT TATAAGCTTGACATAGTACAGCTGGACGGAAATTCTTCCCAATATCGACTGATCA ATTGCGACACATCCGTGATAACCCAGGCGTGCCCCAAAGTTAGTTTCGACCCCAT TCCAATACACTACTGCGCTCCTGCTGGTTACGCTATACTCAAGTGCAATAACAAG ACCTTTACTGGAACGGGTCCATGTAACAATGTGTCTACCGTCCAATGCACACACG GAATAAAACCCGTAGTTAGCACGCAACTGCTGCTGAACGGCTCACTTGCGGAGG GTGAGATCATTATACGGAGCGAAAACATCACGAAGAATGTAAAAACCATCATCGAttorney Docket: 2933311.091.WO1 DU8448PCT TTCACCTGAATGAGAGTGTGAAAATAGAGTGCACCCGACCTAACAATAAGACGA GAACCCTCATCCTCATCGGACCAGGCCAATGGTTCTACGCTACGGGAGACATAAT CGGCGACATCAAGATGGCACACTGCAACATTTCAGAATCCAAATGGAATGAGAC CCTCCAGAGAGTTTCTAAAAAACTCAAAGAATACTTCCCTCACAAGAATATTACG TTTCAACCGTCAAGTGGTGGCGACCTCGAAATAACTACTCACTCTTTCAATTGTG GCGGAGAATTCTTTTATTGCAACACCAGTAGTCTCTTCAATAGAACGTACATGGC TAATAGCACCGATATGGCTAATTCTACCGAAACGAATTCCACTCGGACTATCACC ATTCAGTGCCGGATTAAACAGATTATAAACATGTGGCAAGAGGTGGGTCGAGCA ATGTATGCTCCCCCAATCGCGGGAAACATCACTTGTATAAGTAACATTACTGGCC TGCTGTTGACCCGGGATTACGGGAAGAATAACACTGAAACATTTCGACCAGGAG GTGGAAATATGAAAGACAATTGGCGCAGCGAGCTCTACAAGTATAAAGTAGTAA AGATTGAGCCATTGGGCGTGGCGCCTACTCGCTGCAAGAGAAGGGTAGTAGGCC GGAGAAGGCGGCGGCGCGCCGTGGGAATAGGAGCAGTTAGCTTGGGATTTCTGG GAGCCGCCGGGTCTACAATGGGCGCTGCGTCAATGACACTTACTGTACAGGCAC GCAATCTCCTGAGCGGTATAGTGCAGCAACAAAGTAATTTGCTTCGCGCGCCAGA ACCGCAGCAACACTTGCTTAAGCCGCCGCACTGGGGCATAAAACAGTTGCAAGC AAGGGTACTCGCGGTCGAACACTACTTGCGAGACCAGCAACTTCTGGGCATTTGG GGTTGTAGTGGCAAACTCATCTGTTGCACGAACGTACCGTGGAATAGCTCATGGT CCAACCGCAATCTGTCCGAGATTTGGGACAATATGACGTGGCTCCAGTGGGACA AAGAGATCAGTAATTATACGCAGATCATTTACGGTCTGTTGGAGGAAAGTCAGA ACCAGCAGGAAAAAAATGAGCAAGATCTTCTGGCATTGGAT

[0350] HV1304322: CH505pUCA_I130H CstrandOPT_MPER656_gp150_755 (AA) (SEQ ID NO: 22)

[0351] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLHCTNYAPKLRGMMRGEIKNCSFNATTELRDKRR KEYALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCN NKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLN ESVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRPAttorney Docket: 2933311.091.WO1 DU8448PCT GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQELLELDKWASLWNWFDITNWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGISPLSLQTLTPNPREPDRLRGIEEEGGEQDRDRSIRLVSGF LPI

[0352] HV1304323: CH505pUCA_I130H CstrandOPT_gp150_755 (AA) (SEQ ID NO: 23)

[0353] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLHCTNYAPKLRGMMRGEIKNCSFNATTELRDKRR KEYALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCN NKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLN ESVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKL KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVSLGFL GAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIKQLQAR VLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKE ISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFIMIVGGL IGLRIVFAVLSIVNRVRQGISPLSLQTLTPNPREPDRLRGIEEEGGEQDRDRSIRLVSGF LPI

[0354] HV1304324: CH505pUCA_I130H CstrandOPT_eFP_MPER656_gp150_755 (AA) (SEQ ID NO: 24)

[0355] MRPTWAWWLFLVLLLALWAPARGAENLWVTVYYGVPVWKEAKTTL FCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLKNVTENFNMWKNDMVDQMH EDVISLWDQSLKPCVKLTPLCVTLHCTNYAPKLRGMMRGEIKNCSFNATTELRDKRR KEYALFYKLDIVQLDGNSSQYRLINCDTSVITQACPKVSFDPIPIHYCAPAGYAILKCN NKTFTGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENITKNVKTIIVHLN ESVKIECTRPNNKTRTLILIGPGQWFYATGDIIGDIKMAHCNISESKWNETLQRVSKKLAttorney Docket: 2933311.091.WO1 DU8448PCT KEYFPHKNITFQPSSGGDLEITTHSFNCGGEFFYCNTSSLFNRTYMANSTDMANSTET NSTRTITIQCRIKQIINMWQEVGRAMYAPPIAGNITCISNITGLLLTRDYGKNNTETFRP GGGNMKDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVGIGAVGIGAV SLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEPQQHLLKPPHWGIK QLQARVLAVEHYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWL QWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALDSWNSLWNWFSITKWLWYIKIFI MIVGGLIGLRIVFAVLSIVNRVRQGISPLSLQTLTPNPREPDRLRGIEEEGGEQDRDRSI RLVSGFLPI

[0356] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0357] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0358] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ suchAttorney Docket: 2933311.091.WO1 DU8448PCT variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0359] References

[0360] 1. B. F. Haynes et al., Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies. Nat Rev Immunol 23, 142-158 (2023).

[0361] 2. H. X. Liao et al., Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469-476 (2013).

[0362] 3. C. C. LaBranche et al., Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies. PLoS Pathog 15, e1008026 (2019).

[0363] 4. K. O. Saunders et al., Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques. Cell 187, 79-94 e24 (2024).

[0364] 5. K. O. Saunders et al., Targeted selection of HIV-specific antibody mutations by engineering B cell maturation. Science 366, (2019).

[0365] 6. M. Bonsignori et al., Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies. Sci Transl Med 9, (2017).

[0366] 7. N. T. Freund et al., Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller. Sci Transl Med 9, (2017).

[0367] 8. C. A. Simonich et al., HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant. Cell 166, 77-87 (2016).

[0368] 9. J. M. Steichen et al., A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses. Science 366, (2019).

[0369] 10. N. A. Doria-Rose et al., Developmental pathway for potent V1V2- directed HIV-neutralizing antibodies. Nature 509, 55-62 (2014).

[0370] 11. J. Gorman et al., Structures of HIV-1 Env V1V2 with broadly neutralizing antibodies reveal commonalities that enable vaccine design. Nat Struct Mol Biol 23, 81-90 (2016).Attorney Docket: 2933311.091.WO1 DU8448PCT

[0371] 12. J. R. Willis et al., Human immunoglobulin repertoire analysis guides design of vaccine priming immunogens targeting HIV V2-apex broadly neutralizing antibody precursors. Immunity 55, 2149-2167 e2149 (2022).

[0372] 13. D. Wrapp et al., Structure-Based Stabilization of SOSIP Env Enhances Recombinant Ectodomain Durability and Yield. J Virol 97, e0167322 (2023).

[0373] 14. M. Bonsignori et al., Two distinct broadly neutralizing antibody specificities of different clonal lineages in a single HIV-1-infected donor: implications for vaccine design. J Virol 86, 4688-4692 (2012).

Claims

Attorney Docket: 2933311.091.WO1 DU8448PCT What is claimed is:

1. A recombinant HIV-1 envelope comprising all the consecutive amino acids after the signal peptide of SEQ ID NOS: 1-5 and 21-24.

2. A composition comprising any one of the envelopes of claim 1 and a carrier.

3. The composition of claim 2, wherein the envelope is a protomer comprised in a trimer.

4. The composition of claim 3, wherein the envelope is comprised in a stable trimer.

5. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the envelopes of claim 1.

6. The composition of claim 5, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

7. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the trimers of one of claims 3 or 4.

8. The composition of claim 7, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

9. The composition of claim 8, wherein the nanoparticle comprises multimers of trimers.

10. The composition of claim 8, wherein the nanoparticle comprises 1-8 trimers.

11. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant envelope compositions of any one of claims 1-10 and 21-24, in an amount sufficient to induce an immune response.

12. The method of claim 11, wherein the composition is administered as a prime.

13. The method of claim 11, wherein the composition is administered as a boost.

14. A nucleic acid encoding any of the envelopes of claim 1.

15. A nucleic acid sequence comprising SEQ ID NOs 6-10, and 25.

16. The nucleic acid of one of claims 14 or 15, wherein the nucleic acid is a mRNA.

17. The nucleic acid of claim 16, wherein the mRNA is encapsulated in a lipid nanoparticle.

18. A composition comprising the nucleic acid of any one of claims 14-17 and a carrier.

19. The composition of any one of claims 2-10 or 18 further comprising an adjuvant.

20. The nucleic acid of any one of claims 14-17 or the composition of claim 18 wherein the nucleic acid is operably linked to a promoter, and optionally wherein the nucleic acid is inserted in an expression vector.Attorney Docket: 2933311.091.WO1 DU8448PCT 21. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the nucleic acids of any one of claims 14-17 or 20.

22. The composition of claim 21, wherein the nucleic acid is a mRNA.

23. The composition of one of claims 21 or 22, wherein the nanoparticle is a lipid nanoparticle.

24. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising the nucleic acid of any one of claims 14-17 or 20 or the composition of any one of claims 18-20 in an amount sufficient to induce an immune response.

25. The method of claim 24, further comprising administering an agent which modulates host immune tolerance.

26. The method of any of one of claims 24 or 25, wherein the nucleic acid administered is an mRNA.

27. The method of any of any one of claims 24-26, wherein the nucleic acid is encapsulated in a lipid nanoparticle.

28. The method of any one of claims 24-27, further comprising administering one or more additional HIV-1 immunogens to induce a T cell response.

29. The method of claim 24, wherein the composition is administered as a prime.

30. The method of claim 24, wherein the composition is administered as a boost.

31. A method of inducing an immune response comprising administering an immunogenic composition comprising a prime immunogen comprising all the consecutive amino acids after the signal peptide of SEQ ID NOs: 1-5 and 21-24, followed by at least one boost immunogen from SEQ ID NOs: 1-5 and 21-24, wherein the boost immunogens are administered in an amount sufficient to induce an immune response.

32. The method of claim 31, wherein the prime or boost immunogen is administered as a nanoparticle.

33. The method of claims 31, wherein the nanoparticle is a ferritin nanoparticle.

34. The method of claims 31, wherein the prime or boost immunogen is administered as an mRNA-LNP formulation.

35. An immunogenic composition or composition of any of any one of claims 2-10, 18, 19, or 21-23, wherein the composition comprises at least two different HIV-1 envelope sequences or nucleic acids encoding a HIV-1 envelope, or a combination thereof.Attorney Docket: 2933311.091.WO1 DU8448PCT 36. An immunogenic composition comprising a first immunogen and a second immunogen, wherein the first immunogen is an HIV-1 envelope comprising all the consecutive amino acids after the signal peptide of SEQ ID NOs: 1-5 and 21-24, or a nucleic acid sequence comprising SEQ ID NOs: 6-10 and 25, and wherein the second immunogen is a different HIV-1 envelope comprising all the consecutive amino acids after the signal peptide of SEQ ID NOs: 1-5 and 21-24, or a nucleic acid of SEQ ID NOs: 6-10 and 25.

37. The immunogenic composition of claim 36, wherein at least one of the first immunogen and the second immunogen is a recombinant HIV-1 envelope protein sequence.

38. The immunogenic composition of claim 37, wherein the first immunogen and the second immunogen are both a recombinant HIV-1 envelope protein sequence.

39. The immunogenic composition of claim 36, wherein at least one of the first immunogen and the second immunogen is a nucleic acid.

40. The immunogenic composition of claim 39, wherein the first immunogen and the second immunogen are both a nucleic acid.

41. The immunogenic composition of claim 39 or 40, wherein the nucleic acid is an mRNA.

42. The immunogenic composition of claim 41, wherein the mRNA is encapsulated in an LNP.

43. The immunogenic composition of any one of claims 36-42, further comprising one or more additional immunogens, wherein the one or more additional immunogens are different than the first and second immunogens.

44. The immunogenic composition of any one of claims 36-42, wherein the composition comprises a carrier.

45. The immunogenic composition of any one of claims 36-42, wherein the composition further comprises an adjuvant.

46. A method of inducing an immune response in a subject comprising administering the immunogenic composition according to any one of claims 36-45 in an amount sufficient to induce an immune response.

47. The method of claim 46, further comprising administering an agent which modulates host immune tolerance.Attorney Docket: 2933311.091.WO1 DU8448PCT 48. A recombinant trimer comprising three identical protomers of an envelope polypeptide comprising all the consecutive amino acids after the signal peptide of SEQ ID NOs: 1-5 and 21-24 or the nucleic acid of SEQ ID NOs: 6-10 and 25.

49. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

1.

50. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

2.

51. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

3.

52. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

4.

53. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

5.

54. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

21.

55. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

22.

56. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

23.

57. The recombinant trimer of claim 48, wherein the envelope polypeptide comprises all the consecutive amino acids after the signal peptide of SEQ ID NO:

24.

58. An immunogenic composition comprising the recombinant trimer of any one of claims 48-53 and a carrier.

59. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the trimers of claims 48-53.

60. The composition of claim 55, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

61. The composition of claim 55, wherein the nanoparticle comprises multimers of trimers.

62. The composition of claim 55, wherein the nanoparticle comprises 1-8 trimers.

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