Recombinant HIV-1 env immunogens for eliciting fusion peptide directed antibodies

Abstract

The invention is directed to modified recombinant HIV-1 envelopes comprising one or more modified recombinant HIV-1 fusion proteins, 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: 2933311-097-WO1 DU8523PCT RECOMBINANT HIV-1 ENV IMMUNOGENS FOR ELICITING FUSION PEPTIDE DIRECTED ANTIBODIES REFERENCE TO RELATED APPLICATIONS

[0001] This International Patent Application claims the benefit of U.S. Provisional Patent Application No.63 / 663,639, filed on June 24, 2024, which is hereby incorporated by reference for all purposes as if fully set forth herein. STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant no. R01AI145687 awarded by the NIH, NIGMS, U54AI170752 awarded by the NIH, NIAID, and UM1 AI44371. The government has certain rights in the invention. TECHNICAL FIELD

[0003] 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 modified 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

[0004] The development of a safe and effective HIV-1 vaccine is one of the highest priorities of the scientific community working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients, ART is not routinely available in developing countries. SUMMARY OF THE INVENTION

[0005] 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 (B. F. Haynes et al., Strategies for HIV-1 vaccines that induce broadlyAttorney Docket: 2933311-097-WO1 DU8523PCT neutralizing antibodies. Nat Rev Immunol 23, 142-158 (2023); H. X. Liao et al., Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469-476 (2013)).

[0006] 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), can be 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 (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); K. O. Saunders et al., Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques. Cell 187, 79-94 e24 (2024)). Another such UCA-targeting immunogen is the V1swap Env, which is predominantly derived from the CH84810.17 Env (K. O. Saunders et al., Targeted selection of HIV-specific antibody mutations by engineering B cell maturation. Science 366, (2019)), but has been 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 (M. Bonsignori et al., Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies. Sci Transl Med 9, (2017); 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); C. A. Simonich et al., HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant. Cell 166, 77-87 (2016); J. M. Steichen et al., A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses. Science 366, (2019)). Similar work has been pursued to develop immunogens which are capable of binding to UCAs that can go on to become V2Attorney Docket: 2933311-097-WO1 DU8523PCT 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 (N. A. Doria-Rose et al., Developmental pathway for potent V1V2-directed HIV- neutralizing antibodies. Nature 509, 55-62 (2014); 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); 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)).

[0007] In certain embodiments, the invention provides compositions and methods for induction of an immune response, for example cross-reactive (broadly) neutralizing (bn) Ab induction.

[0008] In certain aspects the invention provides a recombinant protein or nucleic acid encoding a modified recombinant HIV-1 fusion protein as described in SEQ ID NOS: 3-4, and 11-12. In certain aspects, the invention provides a selection of recombinant HIV-1 envelopes comprising one or more modified recombinant HIV-1 fusion proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins for use as a prime immunogen in methods to induce HIV-1 neutralizing antibodies.

[0009] In certain embodiments, the invention provides a recombinant HIV-1 envelope protein or nucleic acid encoding a recombinant HIV-1 envelope protein comprising one or more modified recombinant HIV-1 fusion proteins as described in SEQ ID NOS: 3-4, and 11- 12.

[0010] In certain aspects, the invention provides a recombinant HIV-1 envelope sequence or amino acid encoding a recombinant protein HIV-1 envelope sequence comprising one or more modified recombinant HIV-1 fusion proteins (SEQ ID NOS: 3, 11). In certain aspects, the invention provides a recombinant HIV-1 envelope sequence or nucleic acid encoding aAttorney Docket: 2933311-097-WO1 DU8523PCT recombinant protein HIV-1 envelope sequence comprising one or more modified recombinant HIV-1 fusion proteins modifications (SEQ ID NOS: 4, 12).

[0011] In certain embodiments, the compositions contemplate nucleic acid, as DNA and / or RNA, or proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins of the invention.

[0014] In certain embodiments, the compositions and methods employ a recombinant HIV-1 envelope comprising one or more modified recombinant HIV-1 fusion proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins 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 gp140, and gp41, comprising one or more modified recombinant HIV-1 fusion proteins or N- terminal deletion variants thereof as 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 comprising one or more modified recombinant HIV-1 fusion proteins are multimerized, i.e., multimeric display formats of display here, such as VLPs, nanoparticles, etc. 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. One, but not the only example, of such a nanoparticle would be fusion peptide monomers attached to aAttorney Docket: 2933311-097-WO1 DU8523PCT bacterial ferritin or other type of nanoparticle (See, for example, US Provisional Patent Application No.63 / 540,484). In certain embodiments, the compositions comprise envelopes comprising one or more modified recombinant HIV-1 fusion proteins, 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. In some embodiments, epitopes here can be exposed when there is Env opening.

[0016] In some embodiments, the present invention also provides methods for increasing the immunogenicity of a recombinant HIV-1 envelope comprising insertion of one or more amino acids to the N-terminal portion of the HIV-1 fusion protein portion of the HIV-1 envelope. In some embodiments, the fusion protein portion has two, three, four, or more amino acids inserted. In some embodiments, the amino acids inserted into the fusion protein portion can be AVGIG (SEQ ID NO: 10). In some embodiments, this amino acid motif can be repeated. In some embodiments, this amino acid motif can be altered at each site without limitation to incorporate sequence diversity of circulating HIV-1 isolates. For example, the fusion protein portion can be AVGIGVF (SEQ ID NO: 6). In some embodiments, the fusion protein portion can be AVGIGAVGIGAVF (SEQ ID NO: 8).

[0017] 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.

[0018] In certain embodiments, the envelopes of the present invention are designed to form a stable trimer. In certain embodiments envelope protomers can form a trimer which is not a SOSIP timer. In certain embodiments, the trimer is a SOSIP based trimer wherein each protomer comprises additional modifications. In certain embodiments, the SOSIP-based trimer comprises a BG505.SOSIP trimer or portion thereof. In certain embodiments, envelopeAttorney Docket: 2933311-097-WO1 DU8523PCT trimers are recombinantly produced. In certain embodiments, envelope trimers can be purified from cellular recombinant fractions by antibody binding and reconstituted in lipid comprising formulations. See, for example, WO2015 / 127108, published August 27, 2015, titled “Trimeric HIV-1 envelope compositions 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.

[0019] In certain embodiments, the envelope can be 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 which 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 composition 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 can include TLR7 / 8 ligands, Gardiquimod, Imiquimod and R848 (resiquimod). A example 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).

[0020] In non-limiting embodiments, the adjuvant can be an 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 embodiments include LNPs without polyethylene glycol.

[0021] In certain aspects the invention provides a cell comprising a nucleic acid encoding any one of the envelopes comprising one or more modified recombinant HIV-1 fusionAttorney Docket: 2933311-097-WO1 DU8523PCT proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins of the invention suitable for recombinant expression. In certain aspects, the invention provides a stable pool of cells encoding any one of the envelopes comprising one or more modified recombinant HIV-1 fusion proteins of the invention suitable for recombinant expression.

[0022] In certain aspects the invention provides nucleic acids encoding HIV-1 envelopes comprising one or more modified recombinant HIV-1 fusion proteins 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.

[0023] In certain embodiments, the compositions for use in immunization further comprise an adjuvant.

[0024] In certain embodiments, wherein the compositions comprise a nucleic acid, the nucleic acid is operably linked to a promoter, and could be inserted into an expression vector.

[0025] In one aspect the invention provides a composition for a prime boost immunization regimen comprising any of envelopes comprising one or more modified recombinant HIV-1 fusion proteins described herein, or any combination thereof wherein the envelope comprising one or more modified recombinant HIV-1 fusion proteins is a prime or boost immunogen. In certain embodiments, the composition for a prime boost immunization regimen comprises one or more envelopes comprising one or more modified recombinant HIV-1 fusion proteins described herein, wherein the polypeptide is a non-naturally occurring protomer designed to form an envelope trimer, wherein the envelope comprising one or more modified recombinant HIV-1 fusion proteins 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 comprising one or more modified recombinant HIV-1 fusion proteins of the invention. In one aspect the invention provides a composition for a prime immunization comprising one or more envelopes comprising one or more modified recombinant HIV-1 fusion proteins of the invention.

[0026] In certain aspects the invention provides methods of inducing an immune response in a subject, comprising administering a composition comprising a polypeptide and / or anyAttorney Docket: 2933311-097-WO1 DU8523PCT suitable form of a nucleic acid(s) encoding an HIV-1 envelope(s) comprising one or more modified recombinant HIV-1 fusion proteins in an amount sufficient to induce an immune response.

[0027] 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.

[0028] 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.

[0029] In certain aspects, the invention provides a composition comprising any one of the inventive envelopes comprising one or more modified recombinant HIV-1 fusion proteins or nucleic acid sequences encoding the same.

[0030] In certain aspects, the invention provides compositions comprising a nanoparticle which comprises any one of the envelopes comprising one or more modified recombinant HIV-1 fusion proteins of the invention.

[0031] In certain embodiments, the nanoparticle can be a ferritin self-assembling nanoparticle.

[0032] 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 comprising one or more modified recombinant HIV-1 fusion proteins 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.

[0033] In certain aspects, the invention provides a composition comprising a plurality of nanoparticles comprising a plurality of the envelopes / trimers comprising one or more modified recombinant HIV-1 fusion proteins of the invention. In non-limiting embodiments, the envelopes / trimers comprising one or more modified recombinant HIV-1 fusion proteinsAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0034] 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.

[0035] In some aspects, the invention provides a recombinant HIV-1 extended fusion protein encoded by SEQ ID NOS: 3, 11. In some embodiments, the invention provides an immunogenic composition comprising the recombinant protein and a carrier, wherein the trimer comprises three identical protomers of an HIV-1 envelope comprising one or more modified recombinant HIV-1 fusion proteins, or nucleic acids encoding the fusion proteins, listed in SEQ ID NOS: 3, 4, 11, 12. In some examples, the nucleic acid sequence that encodes SEQ ID NOS: 4 and 12 can be different than SEQ ID NOS: 3 and 11, because of degeneracy of the genetic code.

[0036] 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.

[0037] In some aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises an envelope, wherein the extended fusion protein, or nucleic acid encoding the fusion protein, is selected from SEQ ID NOS: 3 or 4 or any combination thereof. In some embodiments, the compositions comprise two, three, four or more different immunogens. In non-limiting embodiments the different immunogens are selected from the various envelope designs described herein.

[0038] In some embodiments, the nanoparticle of the composition is a ferritin self- assembling nanoparticle.

[0039] 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 extended fusion protein polypeptide from SEQ ID NOS: 4 or 12.

[0040] In some embodiments, the nanoparticle of the composition comprises multiples of fusion proteins.Attorney Docket: 2933311-097-WO1 DU8523PCT

[0041] In some embodiments, the nanoparticle of the composition comprises 1-8 trimers.

[0042] 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 fusion proteins or compositions described herein. In some embodiments the methods comprise administering two, three, four or more different immunogens. In some embodiments, the different immunogenicity target lineage members of VRC34 and other fusion peptide targeting antibodies. In some embodiments, the different immunogenicity target the VRC34 lineage UCA or intermediate antibodies. In non-limiting embodiments the different immunogens are selected from the fusion protein designs described herein at SEQ ID NOS: 4 and 12.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 first composition as a prime and administering the second composition as one or more boosts. In some embodiments, the first composition and the second composition are different.

[0047] In some aspects, the invention provides a nucleic acid encoding any of the recombinant fusion proteins described herein. In some embodiments, the invention provides a composition comprising the nucleic acid and a carrier.Attorney Docket: 2933311-097-WO1 DU8523PCT

[0048] 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 fusion proteins described herein. In some embodiments, the immunogenic composition further comprises a carrier.

[0049] In certain aspects, the invention provides an immunogenic composition or composition, wherein the composition comprises at least two different HIV-1 fusion protein polypeptides or nucleic acids encoding a recombinant HIV-1 fusion protein polypeptide, or a combination thereof.

[0050] 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 fusion protein polypeptide SEQ ID NO: 4 or 12, or encoded by a nucleic acid encoding said recombinant HIV-1 fusion protein polypeptide (SEQ ID NOS: 3 or 11), and wherein the second immunogen is a different recombinant HIV-1 fusion protein polypeptide (SEQ ID NOS: 4, 12) or a nucleic acid encoding said different recombinant HIV- 1 fusion protein polypeptide (SEQ ID NOS: 3, 11). 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 comprising administering an agent which modulates host immune tolerance.

[0051] 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.

[0052] In certain embodiments, the HIV-1 envelopes are in the form of a recombinant HIV-1 envelope polypeptide 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,Attorney Docket: 2933311-097-WO1 DU8523PCT the composition comprises a carrier. In certain embodiments, the composition further comprises an adjuvant.

[0053] In certain embodiments, the present invention provides an anti-HIV antibody which targets the VRC34 lineage UCA. In certain embodiments, the anti-HIV antibody is the VRC34.01 antibody with its glycan N611 binding site deleted (termed “VRC34.01_mut1”). In certain embodiments, the light chain of the VRC34.01_mut1 has the following amino acid substitution: Y94A (SEQ ID NOS: 13, 14). In certain embodiments the heavy chain of the VRC34.01_mut1 has the following amino acid substitutions: D56A, Y68A (SEQ ID NOS: 15, 16). BRIEF DESCRIPTION OF THE FIGURES

[0054] Figure 1 shows an example glycan shielded HIV-1 fusion peptide. The HIV-1 BG505 SOSIP Env is shown in transparent surface representation with underlying cartoon; gp120 is colored grey and gp41 is colored black. The fusion peptide is shown in yellow spheres. The surrounding glycans are shown as spheres, and colored pink: N88, brown; N611, green: N637.

[0055] Figures 2A-C is an illustration of an example extended fusion protein concept of the present invention. The BG505 SOSIP Env is shown colored similarly as in Figure 1. Extended fusion peptide is shown in blue.2A shows a structural representation.2B shows a schematic illustration of the of the eFP peptide construct design.2C shows a schematic illustration of the of the eFP peptide construct design as it will be expressed, indicating the furin cleavage releasing the N terminal eFP end of gp41 that is essential to its antigenicity and antibody binding.

[0056] Figures 3A-3D show examples of extended fusion protein peptides resembling wild type SOSIP Env biochemically.3A is an SDS Page gel.3B shows the results from size- exclusion chromatography.3C depicts the results from differential scanning fluorimetry.3D is a photograph of negative stain electron microscopy showing well folded Env trimers.

[0057] Figure 4 depicts example binding of BG505.SOSIP.eFP to VRC34.01. Prototype FP-directed antibody VRC34.01 was captured on an anti-human Fc chip. BG505 SOSIP produced in 293F cells (complex glycans) (cyan), or BG505 SOSIP produced in GnT1- cells (high mannose glycans) (blue), or BG505 SOSIP eFP produced in 293F cells (complexAttorney Docket: 2933311-097-WO1 DU8523PCT glycans) (black), or BG505 SOSIP eFP produced in GnT1- cells (high mannose glycans) (brown) was flowed over the VRC34.01 surface.

[0058] Figure 5 shows an example cryo-EM structure of BG505.SOSIP.eFP bound to VRC34.01.

[0059] Figures 6A-6B depicts an example of the role of the N611 glycan in VRC34.01 binding to the eFP.6A shows eFP binding.6B shows the eFP construct binding with the VRC34.01 lacking N611 binding site residues.

[0060] Figures 7A-7C shows a zoomed-in view of the VRC34.01 binding interface of BG505.SOSIP.eFP. Panel A shows the VRC34.01 heavy chain in orange, and the light chain in yellow. The FP is colored red and the eFP is colored blue. Glycan 611 is colored green. Panel B shows details of the contact of glycan 611 with VRC34.01. Residues in VRC34.01 that are contacting glycan 611 are shown in stick representation and are labeled. Panel C shows details of the contacts between the FP extension (eFP) and the VRC34.01 light chain. Residues in VRC34.01 that are contacting the engineered extended FP are shown in stick representation and are labeled.

[0061] Figures 8A-8B shows that the VRC34.01 residues that are in contact with glycan 611 and the engineered FP extension (eFP) are mutated from the germline residues during VRC34.01 affinity maturation. Panel A shows an alignment of amino acid sequences of VRC34.01 heavy chain, its inferred germline and affinity maturation intermediates. The positions that are involved in glycan 611 binding are highlighted and labeled. Panel B shows an alignment of amino acid sequences of VRC34.01 light chain, its inferred germline and affinity maturation intermediates. The positions that are involved in eFP binding are highlighted and labeled. The VRC34.01 lineage was described in the following publication Cell Host & Microb.27, 531–543, 8, 2020.

[0062] Figure 9 shows a schematic of the Env.eFP.mut1 construct (also called Env.FP1_mut1), where the native FP (Site 1) is mutated.

[0063] Figures 10A-10B shows an example of how the eFP (site 2) starts the interaction with VRC34.01 using N611 (10A) before moving to native FP (site 1) and engaging with N88 glycan (10B).

[0064] Figures 11A-B. Figure 11 depicts an example of the cryo-EM structures of BG505.SOSIP.eFP.mut1 bound to VRC34.01. Panel A shows the major population observedAttorney Docket: 2933311-097-WO1 DU8523PCT in the cryo-EM dataset with VRC34.01 Fab bound in a downward orientation that is distinct from orientation of VRC34.01 Fab binding to WT Env, indicating that the VRC34.01 Fab was engaging the FP extension (i.e., Site 2). Panel B shows a minor population observed in the cryo-EM dataset that had VRC34.01 bound in a downward orientation at one of the three protomers, and in the other two protomers, VRC34.01 was bound similar to its orientation when bound to the native FP (i.e., Site 1). Thus, the engineered Site 2 extension in the Env.eFP construct is capable of engaging with FP-directed antibodies.

[0065] Figure 12 depicts an example of the amino acid construct of the present invention. HIV-1 Env expresses as a single polypeptide chain (gp160) and gets processed into two subunits, gp120 and gp41, which together form a heterotrimeric complex. The Env ectodomain (gp140) is truncated at 664 amino acid position and mutations were introduced to form a stable disulfide bridge between A501C of gp120 and T605C of gp41 (SOS), gp41 helix extension is abolished by introducing helix breaker, a proline residue at 559th position (I559P) and termed as BG505.SOSIP trimers. A furin cleavage site (6R) introduced between the gp120 and gp41 which gets proteolytically cleaved by Furin upon folding. This process leaves the FP as N-terminal region of gp41. Tandem repeats of the FP sequence “AVGIG” (SEQ ID NO: 10) were introduced to extend the FP in a recombinant BG505.SOSIP Env.

[0066] Figure 13 shows an example of binding assays by SPR to characterize the binding of WT and engineered Envs to VRC34.01, mutants and lineage members. VRC34.01.mut1 is VRC34.01 with its glycan site deleted. Binding was detected to VRC34_I2 intermediate once the H33P substitution was added in. Tighter binding was observed when the T68Y mutation that was observed in the glycan N611 binding site as added in, suggesting a role for the binding of glycan N611 for early lineage members. Mature VRC34.01 does not depend on glycan N611 for binding, thus there is little effect of the N611A glycan deletion mutation on VRC34.01 binding. The eFP construct (SOSIP.eFP) does depend on glycan N611 binding, thus there is dramatic loss of binding when the glycan binding site is mutated in VRC34.01 (VRC34.mut1).

[0067] Figure 14 shows, without being limited to any one example, an example of a schematic diagram showing a possible mechanism for how an embodiment of the Env.eFP construct engages FP-directed antibodies. In the schematic, VRC34.01 is used as a representative example of an FP-directed antibody.Attorney Docket: 2933311-097-WO1 DU8523PCT

[0068] Figures 15A-D. The example data shows the ability of the Env.eFP construct to undergo receptor-mediated conformational changes. To assess whether the FP extension in the eFP construct impacts the ability of the Env to undergo receptor mediated conformational changes, we performed an SPR-based assay described in a recent publication: nature.com / articles / s41467-025-59721-2. Binding was measured of the SOSIP and SOSIP.eFP to VRC34.01 and to 17b at different time points post CD4 or CD4 / 17b Fab addition. We observed similar trends of Env opening and FP binding for both constructs, although reduction in VRC34.01 binding was faster for the eFP construct. These data demonstrate that the SOSIP.eFP ectodomain construct is able to undergo CD4-mediated conformational changes.

[0069] Figures 16A-D shows an example structure of pre-fusion, pre-receptor, closed HIV-1 Env (PDB: 5I8H) bound to broadly neutralizing, fusion peptide-directed antibody VRC34.01 (16A). The Env is shown in surface representation with the gp120 subunits colored light gray, and within the gp120 subunits, the V1V2 loop is colored in wheat, V3 loop in olive and the residues contributing to the bridging sheet in the open Env are colored in red. The gp41 subunits are colored black with the fusion peptide colored cyan. The antibody VRC34.01 is shown in ribbon representation bound to the fusion peptide region. 16B shows the structure of pre-fusion, CD4-bound open HIV-1 Env bound to CD4-induced antibody 17b shown in yellow and orange ribbon.16C shows the Surface plasmon based binding (SPR) analysis monitoring fusion peptide burial through the binding of fusion peptide specific antibody VRC34.01 after incubation with sCD4 alone and in combination with a coreceptor mimicking antibody 17b in time dependent manner.16D shows the binding sensorgrams of 17b and VRC 34.01 Fabs measuring Env opening and fusion peptide burial after incubation of Env with sCD4 to establish a relationship between Env opening and fusion peptide burial.

[0070] Figure 17 shows example cryo-EM maps of particle populations 1, 2 and 3 representing distinct Env conformational states after its incubation with sCD4 and 17b Fab for 2 hr, 20 hrs and 3 days of incubation time at 25 °C. To monitor the position of the fusion peptide VRC34.01 Fab was added post above mentioned incubation time 30 minutes before freezing the cryo-EM grids. In this figure, Env components gp120 / gp41 are colored as grey and black while sCD4, 17b and VRC34.01 are colored yellow, orange and blue.Attorney Docket: 2933311-097-WO1 DU8523PCT

[0071] Figures 18A-F. 18A depicts an example of the cryo-EM structure of major particle population present at all incubation times represents a partially open Env conformational state with gp120 subunit in open conformation and gp41 in closed conformation with exposed fusion peptide. Here, a1 shows the formation of four stranded bridging sheet which is shown as red, a2 is the ^0 helix shown as green, a3 shows HR1s with helical extension towards their N terminal, a4 is exposed fusion peptide shown as cyan with N88 glycan as black engaging with VRC34.01 Fab shown in blue.18B-E represents the top- down views showing the Interprotomer distances between three residues (V3 base residue His330gp120, V1V2 base residue Pro124gp120 and CD4-binding site (CD4bs) residue Asp368gp120) for population 1, closed Env (PDB: 5ACO), fully open B41 Env (PDB 5VN3) and partially open BG505 Env (6CM3).18F table shows the calculated distances.

[0072] Figures 19A-B. 19A shows an example population-1after removing VRC34.01 and 17b Fab structure. Here, gp120, gp41 and sCD4 structural components are with green, black, and yellow color. 19B shows the fitting of population 1 structure into the CD4 bound cryo-ET structure of Env (EMD-29294) present on virion surface shown in grey.

[0073] Figures 20A-C. 20A-B show an example of a cryo-EM map of the particle population 2 and 3 with fusion peptide occluded on one and two gp41 protomers. Here, gp120, gp41, VRC34.01, 17b, and sCD4 are colored as grey, black, blue, orange, and yellow. 20C shows the comparison of structural component having relevance for sCD4 induced conformation changers on gp120 and gp41 protomers between population 2 and 3. Here, the bridging sheet and ^0 helix in gp120 subunit is shown as red and green. The fusion peptide and the remaining gp41 subunit are colored as cyan and black.

[0074] Figures 21A-B. 21A shows examples of superimposed cryo-EM maps of population 1 and 2 where the lateral motion of one of the gp120 subunit in population 2 results into the fusion peptide occluding on the associated gp41 protomer.21B shows the alignments of gp120 and gp41 subunits of population 1 with respective subunits from fully open and population 2 Env. Here, red color on aligned structure represents the highest RMSD differences between the aligned structures.

[0075] Figures 22A-E.22A shows the cryo-EM map of BG505 SOSIP Env with sCD4 and VRC34.01 Fab. The red region on one the gp120 protomer represents the bridging sheet. 22B is cryo-EM map of BG505 SOSIP Env with sCD4 and VRC34.01 Fab with bridgingAttorney Docket: 2933311-097-WO1 DU8523PCT sheet formed on two of gp120 protomers. Here, gp120, gp41, sCD4 and VRC34.01 Fab are colored as grey, black, yellow and blue.22C-E represents the top-down views showing the interprotomer distances between adjacent gp120 protomers measured by calculating distances between V3 base residue and CD4-binding site (CD4bs) residue Asp368gp120) for closed Env (PDB: 5ACO), and sCD4 bound Env with one and two gp120 with bridging sheet formed on them. DETAILED DESCRIPTION OF THE INVENTION

[0076] 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 terms 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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%,Attorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0081] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0082] 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

[0083] The development of a safe, highly efficacious prophylactic HIV-1 vaccine is of paramount importance for the control and prevention of HIV-1 infection. A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) (Immunol. Rev.254: 225-244, 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not reproducibly induced by current vaccines.

[0084] 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.

[0085] A 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. Mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction has been demonstrated (Nature 496: 469, 2013).

[0086] Described herein are nucleic and amino acids sequences of HIV-1 envelopes. The sequences for use as immunogens are in any suitable form.

[0087] An HIV-1 envelope can have 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 ofAttorney Docket: 2933311-097-WO1 DU8523PCT envelopes from different strains are different, the bounds and design of these forms are well known and characterized in the art.

[0088] For example, in some embodiments, 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: 8). 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).

[0089] 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).

[0090] 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0091] SEQ ID NO: 2 is the reference sequence for SEQ ID NO: 4.

[0092] In some embodiments, a nucleic acid that encodes the HIV envelope disclosed herein is set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0093] SEQ ID NO: 1 is the reference sequence for SEQ ID NO: 3.

[0094] In some embodiments, any of the HIV recombinant envelopes can bind to the antibody VRC34.01.

[0095] In some embodiments, any of the HIV recombinant envelopes can bind to the antibodies targeting the HIV-1 fusion peptide.

[0096] 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 someAttorney Docket: 2933311-097-WO1 DU8523PCT embodiments, the HIV recombinant envelopes described herein can bind to neutralizing antibodies that recognize gp160.

[0097] 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.

[0098] 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).

[0099] In some embodiments, the HIV recombinant envelopes described herein can bind to autologous neutralizing antibodies (anAbs). Trimers

[0100] HIV-1 envelope trimers and other envelope designs are described.

[0101] Stabilized HIV-1 Env trimer immunogens can show enhanced antigenicity for broadly neutralizing antibodies and may not be 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.

[0102] 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 Fig.1.

[0103] 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 vaccineAttorney Docket: 2933311-097-WO1 DU8523PCT 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).

[0104] 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.

[0105] It is known that CH505 derived soluble trimers can be 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.

[0106] 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.).

[0107] 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.Attorney Docket: 2933311-097-WO1 DU8523PCT Multimeric Envelopes

[0108] Presentation of antigens as particulates can reduce 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 can provide 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.

[0109] To improve the interaction between the naïve B cell receptor and immunogens, envelope designed can be created such that the envelope is presented on particles, e.g., but not limited to nanoparticles. 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 can have 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.

[0110] Any suitable ferritin sequence could be used. In non-limiting embodiments, ferritin sequences are disclosed in US Patent 10,961,283, issued March 30, 2021, incorporated herein by reference.

[0111] Ferritin nanoparticle linkers: The ability to form HIV-1 envelope ferritin nanoparticles can rely 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 can yield nanoparticles for only certain envelopes. Since the ferritin nanoparticle forms in the absence of envelope, the envelope could be stericallyAttorney Docket: 2933311-097-WO1 DU8523PCT 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 critical 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. A linker length that is suitable for formation of envelope nanoparticles when ferritin is appended to most envelopes can be determined. Any suitable linker between the envelope and ferritin could be uses, so long as the fusion protein is expressed and the trimer is formed.

[0112] Another approach to multimerize expression constructs can use 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: 17) or a N-terminal pentaglycine repeat tag 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.

[0113] The invention can provide designs of envelopes and trimer designs wherein the envelope comprises a linker which permits addition of a lipid, such as but not limited to 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 and immobilization. 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,Attorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0114] The lipid modified envelopes and trimers can be formulated as liposomes. Any suitable liposome composition is contemplated.

[0115] The lipid modified and multimerized envelopes and trimers can be formulated as liposomes. Any suitable liposome composition is contemplated.

[0116] The trimer can be incorporated in a nanoparticle, including without limitation any ferritin-based nanoparticle.

[0117] Throughout the application amino acid positions numbers refer to HXB2 numbering.

[0118] 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.

[0119] Modifications of nucleic acids encoding the inventive envelopes may include:

[0120] 5’UTR including aGcATAAAAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAG CATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTT CTGAAAATTTTCACCATTTACGAACGATAGCGCT (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.

[0121] 3’UTR including actagtAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACACCCGA ATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTTGTCCCCCA AAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTCT (SEQ ID NO: 19). Without being bound by theory, this modification is an improved 5' UTR sequence for mRNA stability and half-life from screens. See Messenger RNA-BasedAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0122] poly A (immediately after 3’UTR) includes AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 20). 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).

[0123] 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 follow: 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).

[0124] 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.

[0125] In non-limiting embodiments, sortase A tagged recombinant envelope trimers can be displayed on the surface of sortase A conjugated ferritin nanoparticles.

[0126] In some embodiments, the multimer is a nanoparticle including recombinant envelope trimers including the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, orAttorney Docket: 2933311-097-WO1 DU8523PCT 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 / or 12.

[0127] In some embodiments, a recombinant envelope trimer including the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and / or 12, 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.

[0128] In some embodiments, any of the envelope trimers described herein can be lipid modified as described herein. In some embodiments, the amino acid sequence (SEQ ID No: 4 and / or 12) can be lipid modified as described herein.

[0129] In some embodiments, mRNA encoded by the nucleotide set forth in SEQ ID NO: 3 and / or 11, or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and / or 11, can be displayed and / or encapsulated in a lipid nanoparticle. Compositions

[0130] In certain aspects, the invention provides a selection of HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins as multimerized trimers, as proteins, DNAs, RNAs, or any combination thereof, administered as primes and boosts to elicit immune response. HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins as proteins would be co-administered with nucleic acid vectors containing HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins 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 mosaicAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0131] In some embodiments, the composition can include HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins of SEQ ID NO: 4 and / or 12, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 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.

[0132] 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and / or 11, wherein the lipid nanoparticle includes 1, 2, 3, 4, 5, 5, 6, 7 or 8 recombinant envelope trimers and a carrier and / or adjuvant.

[0133] In some embodiments, the composition includes a ferritin self assembling nanoparticle including the amino acid sequence set forth in SEQ ID NO: 4 and / or 12, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and / or 12, wherein the nanoparticle displays 1, 2, 3, 4, 5, 6, 7 or 8 recombinant envelope trimers Nucleic acids

[0134] In certain aspects the invention can provide 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,Attorney Docket: 2933311-097-WO1 DU8523PCT the invention provides a stable pool of cells encoding any one of the envelopes of the invention suitable for recombinant expression.

[0135] In certain aspects, the invention provides a polypeptide encoding HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins. 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.

[0136] In certain aspects the invention provides a recombinant trimer comprising three identical protomers of the HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins. In certain aspects the invention provides an immunogenic composition comprising the recombinant trimer and a carrier, wherein the trimer comprises three identical protomers of the HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins. In certain aspects the invention provides an immunogenic composition comprising a nucleic acid encoding these recombinant HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins and a carrier.

[0137] 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.

[0138] 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.

[0139] 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 vectorAttorney Docket: 2933311-097-WO1 DU8523PCT 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 invention 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.

[0140] 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. Mutant VRC34.01 HIV Antibody

[0141] In certain embodiments, the present invention provides an anti-HIV antibody. In some embodiments, the anti-HIV antibody can be a mutant antibody. In certain embodiments, the present invention provides an anti-HIV antibody which targets the target the VRC34 lineage UCA. In certain embodiments, the anti-HIV antibody is the VRC34.01 antibody with its glycan N611 binding site deleted (termed “VRC34.01_mut1”). In certain embodiments, the light chain of the VRC34.01_mut1 has the following amino acid substitution: Y94A (SEQ ID NOS: 13, 14). In certain embodiments the heavy chain of the VRC34.01_mut1 has the following amino acid substitutions: D56A, Y68A (SEQ ID NOS: 15, 16).

[0142] In certain aspects, provided are antibodies and fragments of VRC34.01_mut1 comprising VHand VL(as used herein, VHand VLcan also be referred to as VH or VL and VH or VL, respectively) sequences of the antibodies described in SEQ ID NOS: 13-16.

[0143] In certain aspects, the antibodies are recombinant antibodies having an IgG or IgM Fc domain, or a portion thereof.

[0144] In certain aspects, the invention provides a pharmaceutical composition comprising the recombinant antibodies of the invention.Attorney Docket: 2933311-097-WO1 DU8523PCT

[0145] In certain aspects, the invention provides nucleic acids comprising sequences encoding antibodies comprising VH and VL sequences of the inventive antibodies, e.g. from SEQ ID NOS: 13-16. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention. Methods

[0146] 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.

[0147] 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 certain embodiments the nucleic acids are optimized for expression in vectors and / or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus-based vectors, rAdenovirus (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.Attorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0148] 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 can be used herein. 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.

[0149] 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.

[0150] 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.

[0151] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences ofAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0152] 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.

[0153] 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.

[0154] 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; SEQ ID NO: 21) near its 3' end. In 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.

[0155] The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the envelope.Attorney Docket: 2933311-097-WO1 DU8523PCT 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).

[0156] Methods for in vitro transfection of mRNA and detection of envelope expression are known in the art.

[0157] Methods for expression and immunogenicity determination of nucleic acid encoded envelopes are known in the art.

[0158] 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.

[0159] 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).

[0160] 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.

[0161] 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.

[0162] 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 otherAttorney Docket: 2933311-097-WO1 DU8523PCT squalene-based adjuvant, ASOIB, or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen (Leroux- Roels et al., IABS Conference, 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.

[0163] 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.

[0164] There are various host mechanisms that can control bnAbs. For example, highly somatically mutated antibodies become autoreactive and / or less fit (Immunity 8: 751, 1998; PloS Comp. Biol.6 e1000800, 2010; J. Thoret. Biol.164:37, 1993); Polyreactive / autoreactive naïve B cell receptors (unmutated common ancestors of clonal lineages) can lead to deletion of Ab precursors (Nature 373: 252, 1995; PNAS 107: 181, 2010; J. Immunol.187: 3785, 2011); Abs with long HCDR3 can be limited by tolerance deletion (JI 162: 6060, 1999; JCIAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0165] 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: 14). 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.

[0166] 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 sequences of SEQ ID NOS: 4, 12.

[0167] 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. In some embodiments, the methods of inducing an immune response in a subject include administering an immunogenic composition comprising any one of the of the HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusionAttorney Docket: 2933311-097-WO1 DU8523PCT proteins described herein and / or any of the compositions described herein in an amount sufficient to induce an immune response.

[0168] 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 NOS: 4, 12, 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: 4, 12, in an amount sufficient to induce an immune response in a subject.

[0169] 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 of the HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins 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.

[0170] In some embodiments, the methods of inducing an immune response in a subject include administering an immunogenic composition comprising any one of the of the HIV-1 envelope compositions comprising one or more modified recombinant HIV-1 fusion proteins 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.

[0171] 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 NOS: 4, 12, as a prime and a composition including aAttorney Docket: 2933311-097-WO1 DU8523PCT recombinant envelope including the amino acid sequence set forth in SEQ ID NOS: 4, 12, as a boost.Attorney Docket: 2933311-097-WO1 DU8523PCT EXAMPLES

[0172] 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 – eFP Construct Design

[0173] HIV-1 Env expresses as a single polypeptide chain (gp160) and gets processed into two subunits, gp120 and g41, which together form a heterotrimeric complex. The Env ectodomain (gp140) is truncated at 664 amino acid position and mutations were introduced to form a stable disulfide bridge between A501C of gp120 and T605C of gp41 (SOS), gp41 helix extension is abolished by introducing helix breaker, a proline residue at 559th position (I559P) and termed as BG505.SOSIP trimers. A furin cleavage site (6R) introduced between the gp120 and gp41 which gets proteolytically cleaved by Furin upon folding (Sanders, R. W. et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog 9, e1003618 (2013) doi.org / 10.1371 / journal.ppat.1003618). This process leaves the FP as N-terminal region of gp41. We introduced tandem repeats of the FP sequence “AVGIG” (SEQ ID NO: 10) to extend the FP in a recombinant BG505.SOSIP Env. The new designed construct was named BG505.SOSIP.eFP (Fig.2B-C). EXAMPLE 2 – Envelope Protein Purification

[0174] HIV-1 Env ectodomain constructs were produced and purified as described previously (Henderson, R. et al. Structural basis for breadth development in the HIV-1 V3- glycan targeting DH270 antibody clonal lineage. Nat Commun 14, 2782 (2023) doi.org / 10.1038 / s41467-023-38108-1). All the Envs used in this study were purified from Freestyle 293F GnTI- cells, which were diluted at the time of transfection to 1.5 x 106Attorney Docket: 2933311-097-WO1 DU8523PCT cells / mL. Co-transfection was performed with plasmid DNA (650 µg SOSIP trimer plasmid and 150 µg furin-expressing plasmid per 1L of culture volume) complexed with 293fectin in Opti-MEM. After 6 days, cell cultures were harvested by centrifugation of the cells for 30 minutes at ~3500 rpm on a tabletop centrifuge. The supernatant was filtered through a 0.22 µm filter. Following filtration, the supernatant was passed through a 10 mL PGT145-agarose affinity column buffered with 20 mM PBS, pH 7.5. Following loading and washing, Env trimers were eluted using 3M MgCl2. The eluted Env was concentrated to ~1 mL with a Centricon-70100 kDa filter. After concentrating, Env was filtered through a 0.22 µm filter to remove any aggregates before loading on a Superose-6 Increase 10 / 300 GL size exclusion column (Cytiva) pre-equilibrated in PBS containing 0.02% w / v NaN3 on an AKTA Pure (Cytiva) workstation. The trimeric pool of protein fractions was pooled, concentrated, and flash frozen for long-term storage at -80 °C. Each aliquot was thawed and briefly incubated (roughly 2 minutes) at 37 °C before use. Further quality control on the purified protein was performed by SDS-PAGE, NSEM, and DSF-based stability analysis. EXAMPLE 3 – Antibody Purification

[0175] Antibodies were produced in Expi293F cells and purified using a Protein-A affinity column. Following purification size exclusion chromatography was performed for further purification and buffer exchange antibodies in 20 mM PBS, pH 7.5, 0.02% w / v NaN3. EXAMPLE 4 – Thermostability Assays

[0176] Thermostability of the HIV-1 Env samples was measured using differential scanning fluorimetry (DSF). DSF assay was performed using Tycho NT.6 (NanoTemper Technologies) for all Env variants at 0.1244 mg ml−1 in PBS with 0.02% NaN3and triplicate were performed for reproducibility. Intrinsic fluorescence was recorded at 330 and 350 nm while heating the sample from 35–95 °C at a rate of 30 °C min−1. The ratio of fluorescence (350 / 330 nm) and its first derivative were plotted against temperature to calculate the inflection temperatures (Ti). EXAMPLE 5 – Negative-Stain Electron Microscopy NSEMAttorney Docket: 2933311-097-WO1 DU8523PCT

[0177] A frozen aliquot from -80 °C was thawed at RT in Al block for 5 min. Sample was then diluted to 200 µg / ml with 5 g / dl Glycerol in HBS (20 mM HEPES, 150 mM NaCl pH 7.4) buffer containing 8 mM glutaraldehyde. After 5 min incubation, glutaraldehyde was quenched by adding sufficient 1 M Tris stock, pH 7.4, to give 80 mM final Tris concentration and incubated for 5 min. Quenched sample was applied to a glow-discharged carbon-coated EM grid for 10-12 seconds, then blotted, and stained with 2 g / dL uranyl formate for 1 min, blotted and air-dried. Grids were examined on a Philips EM420 electron microscope operating at 120 kV and nominal magnification of 49,000x, and 13 images were collected on a 76 Mpix CCD camera at 2.4 Å / pixel. Images were analyzed by 2D class averages using standard protocols with Relion 3.0 (Zivanov, J. et al. New tools for automated high- resolution cryo-EM structure determination in RELION-3. Elife 7 (2018) doi.org / 10.7554 / eLife.42166). EXAMPLE 6 – Surface Plasmon Resonance (SPR) Affinity Analysis

[0178] The binding affinity of antibodies with BG505.SOSIP was checked by SPR on T- 200 Biacore system (GE-Healthcare) operating at 25 ^C. The experiment was performed in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P-20) buffer. The binding analysis was performed by immobilizing 200 nM of IgGs (VRC34.01 and VRC 34.01_mut1) after flowing @ 10 µl / min for 60 seconds over the antihuman IgG coated CM5 chip in all flow cell except for the cell used as reference. After preparing the binding surface, the Envs (40nM) were flowed @ 30 µl / min for 120 seconds association time followed by the dissociation time of 120 seconds. The final binding curve was obtained after black correcting the binding sensorgrams in Biacore T-200 evaluation software. Graph pad Prism software was used for data plotting. An example is shown in Fig.4. EXAMPLE 7 – Cryo-EM Sample Preparation, Data Collection and Processing

[0179] For determining the cryo-EM structure of BG505.SOSIP_eFP bound to VRC34.01, purified BG505.SOSIP_eFP trimer were diluted to a final concentration of 1.3 mg / mL in 20 mM Phosphate buffered saline (PBS), pH 7.5 and mixed with five molar excess of VRC34.01 Fab and left for incubation at room temperature for 30 minutes. For determining structures of CD4-triggered BG505.SOSIP trimer, BG505.SOSIP was diluted toAttorney Docket: 2933311-097-WO1 DU8523PCT a concentration of 1.3 mg / mL and incubated with five molar excess of 4D-CD4 and 5-molar excess of 17-b Fab. After mixing, the samples were incubated at room temperature (25 ^C) for different incubation times and VRC34.01 Fab in five molar excess concentration to SOSIP was added 30 minutes before freezing grids. To prevent the interaction of the trimer complexes with the air-water interface during vitrification, the samples were incubated in 0.085 mM n-dodecyl β-D-maltoside (DDM). A 3.5-μL drop of protein was deposited on a Quantifoil-1.2 / 1.3 grid (Electron Microscopy Sciences, PA) that had been glow discharged for 10 s using a PELCO easiGlow Glow Discharge Cleaning System. After a 30 seconds incubation in >95% humidity, excess protein was blotted away for 2.5 s before being plunge frozen into liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids were imaged using a Titan Krios (Thermo Fisher) equipped with a K3 direct detector (Gatan) and operating at 300kV. The data processing was done with the cryoSPARC software (Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290-296 (2017) doi.org / 10.1038 / nmeth.4169) including motion correction of the raw movies. Phenix (Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861-877 (2019) doi.org / 10.1107 / S2059798319011471, and Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D Struct Biol 74, 531-544 (2018) doi.org / 10.1107 / S2059798318006551), Coot (Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010) doi.org / 10.1107 / S0907444910007493), Pymol (Schrodinger, L. The PyMOL Molecular Graphics System (2015), www.pymol.org), and ChimeraX (Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 27, 14-25 (2018) doi.org / 10.1002 / pro.3235) were used for model building and refinement. An example is shown in Fig.5. SEQUENCES

[0180] HV1301379_WT (BG505gp140SOSIP.T332N_WT) (SEQ ID NO: 1)

[0181] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCC TATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCAAttorney Docket: 2933311-097-WO1 DU8523PCT AGTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCT GGAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGAC CGAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCC CCAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAA TATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAA GCCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCACA AACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATG ACCACAGAACTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTG GATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAG GAATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGCTTGTCCTAAAG TGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTG AAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAG TCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAATGG AAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGC CAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTCGGAATTGGGGCC GTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGGGAGCAGCCTCAATGA CTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCGTCCAGCAGCAGTCCA ACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGAAGCTGACCGTGTGGG GCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGCGGTACCTGAGAGACCAttorney Docket: 2933311-097-WO1 DU8523PCT AGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGATTTGTTGCACAAATGT GCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGAGATCTGGGACAATATG ACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACACCCAGATCATCTACGGC CTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGAACAGGACCTGCTGGC ACTGGATTGAGGATCCamino acid translation) (SEQ ID NO: 2)

[0183] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVY SLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFA ILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAK NILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETL GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS TNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAVG IGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTV WGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDN MTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[0184] BG505.SOSIP.eFP (SEQ ID NO: 3)

[0185] GTCGACACGTGTGATCAGATATCGCGGCCGCTCTAGAGCCACCATGCC TATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGAATGCTGGTCGCA AGTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATGGAGTGCCCGTCT GGAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAAGGCCTACGAGAC CGAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCACCGACCCAAACCC CCAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACATGTGGAAAAACAA TATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGATCAGTCCCTGAA GCCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGTACCAATGTCACA AACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGTAGCTTCAACATG ACCACAGAACTGAGAGACAAGAAACAGAAAGTGTACTCCCTGTTTTATAGGCTGAttorney Docket: 2933311-097-WO1 DU8523PCT GATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAACAATTCCAACAAG GAATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGGCTTGTCCTAAAG TGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGCTTCGCCATCCTG AAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTTCCGTGTCTACAG TCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCTGCTGCTGAATGG AAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACATTACCAACAATGC CAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAATTGCACAAGACCC AACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAGGCCTTTTACGCTA CAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATGTGAGTAAGGCAA CCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGGAAACATTTCGGGA ATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACCTGGAGGTCACTAC CCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACACATCTGGCCTGTTTA ATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAATTCAACTGGGTCAAA CGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTAATATGTGGCAGCGG ATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCATTCGCTGCGTGAGCA ATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCACCAACTCTACAACTG AAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTGGAGGTCCGAGCTGT ACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGCACCAACCAGATGCA AGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAGTAGGGATCGGGGCA GTCGGAATTGGGGCCGTGTTCCTGGGATTTCTGGGCGCCGCTGGGAGTACAATGG GAGCAGCCTCAATGACTCTGACCGTGCAGGCCAGGAATCTGCTGAGCGGCATCG TCCAGCAGCAGTCCAACCTGCTGCGCGCTCCTGAAGCACAGCAGCACCTGCTGA AGCTGACCGTGTGGGGCATCAAACAGCTGCAGGCTAGGGTGCTGGCAGTCGAGC GGTACCTGAGAGACCAGCAGCTGCTGGGAATCTGGGGCTGCTCTGGGAAGCTGA TTTGTTGCACAAATGTGCCTTGGAACTCTAGTTGGTCAAATCGCAACCTGAGCGA GATCTGGGACAATATGACTTGGCTGCAGTGGGATAAAGAAATTAGTAACTACAC CCAGATCATCTACGGCCTGCTGGAAGAGTCACAGAATCAGCAGGAGAAGAACGA ACAGGACCTGCTGGCACTGGATTGAGGATCC

[0186] BG505.SOSIP.eFP amino acid translation (SEQ ID NO: 4)Attorney Docket: 2933311-097-WO1 DU8523PCT

[0187] MPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYGVPVWKDAETTLFC ASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDII SLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVY SLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFA ILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEEEVMIRSENITNNAK NILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNVSKATWNETL GKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCNTSGLFNSTWISNTS VQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSNITGLILTRDGGS TNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKRRVVGRRRRRRAV GIGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQH LLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLS EIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLALD

[0188] HIV-1 Fusion Protein Peptide Portion WT (SEQ ID NO: 5)

[0189] GCCGTCGGAATCGGAGCCGTGTTC

[0190] HIV-1 Fusion Protein Peptide Portion WT amino acid translation (SEQ ID NO: 6)

[0191] AVGIGAVF

[0192] Extended HIV-1 Fusion Protein Peptide Portion (SEQ ID NO: 7)

[0193] GCAGTCGGAATTGGGGCCGTCGGAATCGGAGCCGTGTTC

[0194] Extended HIV-1 Fusion Protein Peptide Portion amino acid translation (SEQ ID NO: 8)

[0195] AVGIGAVGIGAVF

[0196] Fusion Protein Extension Sequence (SEQ ID NO: 9)

[0197] GCAGTCGGAATTGGG

[0198] Fusion Protein Extension Sequence amino acid translation (SEQ ID NO: 10)

[0199] AVGIGAttorney Docket: 2933311-097-WO1 DU8523PCT

[0200] BG505gp140SOSIP.T332N_FP1_mut1 (SEQ ID NO: 11)

[0201] ATGCCTATGGGGAGCCTGCAGCCTCTGGCAACCCTGTATCTGCTGGGA ATGCTGGTCGCAAGTGTCCTGGCCGCCGAAAACCTGTGGGTCACCGTGTATTATG GAGTGCCCGTCTGGAAAGATGCTGAAACTACCCTGTTCTGTGCCTCTGATGCTAA GGCCTACGAGACCGAAAAGCACAATGTCTGGGCTACTCATGCATGCGTGCCCAC CGACCCAAACCCCCAGGAGATCCACCTGGAAAATGTGACCGAGGAATTCAACAT GTGGAAAAACAATATGGTGGAGCAGATGCATACAGACATCATTAGCCTGTGGGA TCAGTCCCTGAAGCCCTGCGTCAAACTGACTCCTCTGTGCGTGACCCTGCAGTGT ACCAATGTCACAAACAATATCACCGACGATATGAGGGGCGAGCTGAAGAATTGT AGCTTCAACATGACCACAGAACTGAGAGACAAGAAACAGAAAGTGTACTCCCTG TTTTATAGGCTGGATGTGGTCCAGATCAATGAGAACCAGGGGAATCGGAGCAAC AATTCCAACAAGGAATACAGACTGATCAATTGCAACACTTCCGCCATTACCCAGG CTTGTCCTAAAGTGTCTTTTGAGCCTATCCCAATTCATTATTGCGCCCCAGCTGGC TTCGCCATCCTGAAGTGTAAAGATAAGAAGTTCAACGGAACTGGCCCCTGCCCTT CCGTGTCTACAGTCCAGTGTACTCACGGGATTAAGCCTGTGGTCTCTACACAGCT GCTGCTGAATGGAAGTCTGGCTGAGGAAGAAGTGATGATCCGGAGCGAGAACAT TACCAACAATGCCAAGAATATCCTGGTCCAGTTCAACACACCAGTGCAGATTAAT TGCACAAGACCCAACAATAACACTCGAAAATCTATCCGGATTGGGCCAGGACAG GCCTTTTACGCTACAGGGGACATCATTGGAGATATCAGACAGGCTCACTGTAATG TGAGTAAGGCAACCTGGAACGAGACACTGGGCAAGGTGGTCAAACAGCTGAGG AAACATTTCGGGAATAACACCATCATTCGCTTTGCCAATAGCTCCGGAGGGGACC TGGAGGTCACTACCCACTCCTTCAACTGCGGAGGCGAATTCTTTTACTGTAACAC ATCTGGCCTGTTTAATAGTACATGGATCTCTAACACTAGTGTGCAGGGCAGTAAT TCAACTGGGTCAAACGATAGCATCACCCTGCCATGCCGAATTAAGCAGATCATTA ATATGTGGCAGCGGATCGGCCAGGCAATGTATGCCCCCCCTATCCAGGGGGTCAT TCGCTGCGTGAGCAATATCACCGGACTGATTCTGACACGAGACGGGGGCAGCAC CAACTCTACAACTGAAACATTCCGGCCCGGCGGGGGAGACATGAGAGATAACTG GAGGTCCGAGCTGTACAAGTATAAAGTGGTCAAGATCGAACCTCTGGGAGTGGC ACCAACCAGATGCAAGCGAAGAGTGGTCGGACGAAGGAGGAGGAGGCGAGCAG TCGGAATTGGGGCCGTCGGAATGGGAGCCCTGTTCCTGGGATTTCTGGGCGCCGCAttorney Docket: 2933311-097-WO1 DU8523PCT TGGGAGTACAATGGGAGCAGCCTCAATGACTCTGACCGTGCAGGCCAGGAATCT GCTGAGCGGCATCGTCCAGCAGCAGTCCAACCTGCTGCGCGCTCCTGAAGCACA GCAGCACCTGCTGAAGCTGACCGTGTGGGGCATCAAACAGCTGCAGGCTAGGGT GCTGGCAGTCGAGCGGTACCTGAGAGACCAGCAGCTGCTGGGAATCTGGGGCTG CTCTGGGAAGCTGATTTGTTGCACAAATGTGCCTTGGAACTCTAGTTGGTCAAAT CGCAACCTGAGCGAGATCTGGGACAATATGACTTGGCTGCAGTGGGATAAAGAA ATTAGTAACTACACCCAGATCATCTACGGCCTGCTGGAAGAGTCACAGAATCAG CAGGAGAAGAACGAACAGGACCTGCTGGCACTGGAT

[0202] BG505gp140SOSIP.T332N_FP1_mut1 amino acid translation (SEQ ID NO: 12)

[0203] STRVIRYRGRSRATMPMGSLQPLATLYLLGMLVASVLAAENLWVTVYYG VPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMW KNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFN MTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSAITQACPKVS FEPIPIHYCAPAGFAILKCKDKKFNGTGPCPSVSTVQCTHGIKPVVSTQLLLNGSLAEE EVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQA HCNVSKATWNETLGKVVKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNCGGEFFYCN TSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCV SNITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTRCKR RVVGRRRRRRAVGIGAVGMGALFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQ QSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVERYLRDQQLLGIWGCSGKLICCTN VPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQEKNEQDLLAL D

[0204] VRC34.01_mut1 Light Chain (SEQ ID NO: 13)

[0205] ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTG TACATTCTGACATCCAGTTGACCCAGTCTCCATCCTTCCTTTCTGCCTCTGTAGGA GACAAGGTTACGATCACTTGCCGGGCCAGTCAGGGCGTTCGCAATGAGTTAGCCT GGTATCAGCAAAAACCAGGGAAAGCCCCTAATCTCCTCATCTATTATGCATCCAC TTTGCAAAGTGGGGTCCCGTCAAGATTCAGCGCCACTGGATCTGGGACACACTTC ACTCTCACAGTCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTTCTGTCAACAttorney Docket: 2933311-097-WO1 DU8523PCT ACATGAGCAGTGCCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAACAGCCAGGAAAG CGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCTGAGCAGCACACTGAC ACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACA CCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGAGAATGC

[0206] VRC34.01_mut1 Light Chain (SEQ ID NO: 14) amino acid translation

[0207] MGWSCIILFLVATATGVHSDIQLTQSPSFLSASVGDKVTITCRASQGVRNE LAWYQQKPGKAPNLLIYYASTLQSGVPSRFSATGSGTHFTLTVSSLQPEDFATYFCQ HMSSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNR

[0208] VRC34.01_mut1 Heavy Chain (SEQ ID NO: 15) ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATT CCCAGGAGGTACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCCTCAG TGAAGGTCTCCTGCAGGGCTTTTGGATATACCTTTACCGGCAATGCTCTGCACTG GGTTCGGCAGGCCCCTGGGCAAGGTCTTGAGTGGCTGGGGTGGATCAACCCTCA CAGTGGTGCCACAACCACTTCACAGAAATTTCAGGGCAGGGTCGCCATGACCAG GGACAAGTCCATCAACACAGCCTTTTTGGACGTGACCAGGCTAACATCTGACGAC ACGGGCATATATTATTGTGCGAGAGACAAGTACTATGGTAATGAGGCAGTCGGA ATGGACGTCTGGGGCCAGGGGACCTCGGTCACCGTCTCCTCAGCGTCGACCAAG GGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCCGTGACGGTGTCGTG GAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCC TCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCA CCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCAttorney Docket: 2933311-097-WO1 DU8523PCT CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0209] VRC34.01_mut1 Heavy Chain (SEQ ID NO: 16) amino acid translation MGWSCIILFLVATATGVHSQEVLVQSGAEVKKPGASVKVSCRAFGYTFTGNALHWV RQAPGQGLEWLGWINPHSGATTTSQKFQGRVAMTRDKSINTAFLDVTRLTSDDTGIY

[0210] 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.

[0211] 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 methodsAttorney Docket: 2933311-097-WO1 DU8523PCT 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.

[0212] 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 such 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.

Claims

Attorney Docket: 2933311-097-WO1 DU8523PCT What is claimed is:

1. A recombinant HIV-1 envelope comprising one or more modified recombinant HIV-1 fusion proteins disclosed herein.

2. The recombinant HIV-1 envelope of claim 1, wherein the one or more modified recombinant HIV-1 fusion proteins are encoded by the nucleotide sequence of SEQ ID NO: 3, or comprises the amino acid sequence of SEQ ID NOS: 4 and / or 12.

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

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

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

6. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the envelopes of claim 2.

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

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

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

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

11. The composition of claim 9, wherein the nanoparticle comprises 1-8 trimers.Attorney Docket: 2933311-097-WO1 DU8523PCT 12. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant envelope compositions of claim 2, in an amount sufficient to induce an immune response.

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

14. The method of claim 12, wherein the composition is administered as a boost.

15. A nucleic acid encoding any one of the envelopes of claim 2.

16. A nucleic acid sequence comprising SEQ ID NOS: 3 and / or 11.

17. The nucleic acid of claim 16, wherein the nucleic acid is an mRNA.

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

19. A composition comprising the nucleic acid of claim 15 and a carrier.

20. The composition of any one of claims 3 or 19 further comprising an adjuvant.

21. The nucleic acid of claim 15 or the composition of claim 19 wherein the nucleic acid is operably linked to a promoter, and optionally wherein the nucleic acid is inserted in an expression vector.

22. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the nucleic acids of claim 15.

23. The composition of claim 22, wherein the nucleic acid is an mRNA.Attorney Docket: 2933311-097-WO1 DU8523PCT 24. The composition of claims 22, wherein the nanoparticle is a lipid nanoparticle.

25. A method for increasing the immunogenicity of a recombinant HIV-1 envelope comprising insertion of one or more amino acids to the N-terminal portion of the HIV-1 fusion protein portion of the HIV-1 envelope.

26. The method of claim 25, wherein the fusion protein portion has two, three, four, or more amino acids inserted.

27. The method of claim 25, wherein the amino acids inserted into the fusion protein portion are AVGIG (SEQ ID NO: 10).

28. The method of claim 27, wherein the amino acid sequence AVGIG (SEQ ID NO: 10) is repeated.

29. An anti-HIV antibody and / or fragments of portions thereof, wherein said antibody comprises a VL domain having the amino acid sequence of SEQ ID NO:

14.

30. An anti-HIV antibody and / or fragments of portions thereof, wherein said antibody comprises a VH domain having the amino acid sequence of SEQ ID NO:

16.

31. The anti-HIV antibody and / or fragments of portions thereof of claim 29, wherein said antibody comprises a VL domain having the following amino acid substitution: Y94A.

32. The anti-HIV antibody and / or fragments of portions thereof of claim 31, wherein said antibody comprises a VH domain having the following amino acid substitutions: D56A, Y68A.

33. A recombinant nucleic acid sequence encoding the anti-HIV antibody of claim 29.Attorney Docket: 2933311-097-WO1 DU8523PCT 34. The recombinant nucleic acid sequence of claim 33, having the nucleic acid sequence of SEQ ID NO:

13.

35. A recombinant nucleic acid sequence encoding the anti-HIV antibody of claim 32.

36. The recombinant nucleic acid sequence of claim 35, having the nucleic acid sequence of SEQ ID NO:

15.

37. The anti-HIV antibody or nucleic acid encoding the antibody of any of claims 29-36, wherein said antibodies are recombinant antibodies having an IgG or IgM Fc domain, or a portion thereof.

38. A pharmaceutical composition comprising the recombinant antibodies or nucleic acid encoding the antibody of any of claims 29-36 and a pharmaceutically acceptable carrier.

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