Immunogens and vaccine compositions against HIV

Abstract

Disclosed herein Arc modified HIV-1 Env polypeptides comprising at least one modified hypervariable loop and isolated polynucleotides comprising a nucleotide sequence that encodes the modified HIV-1 Env polypeptides. Also disclosed herein are vaccine or immunogenic compositions for inducing an immune response in a subject against HIV, as well as a method of inducing an immune response against HIV in a subject. Further disclosed herein are methods of identifying an antibody against HIV in a sample.

Description

Attorney Docket No.: HMJ-190-PCT IMMUNOGENS AND VACCINE COMPOSITIONS AGAINST HIV CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 585,686 filed 27 September 2023, the entire contents of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format as a XML file and hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is HMJ-190-PCT_SL.xml and the size of the XML file is 252,148 bytes. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under W81XWH-18-2-0040 awarded by the Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention. FIELD

[0004] This application relates generally to novel human immunodeficiency virus (HIV) immunogens and to methods and compositions related thereto. More particularly, the disclosure relates to compositions and methods for the preparation, production, and administration of isolated novel HIV immunogen nucleic acid and protein sequences suitable, for example, in certain embodiments, as vaccines against HIV and for capturing antibodies against HIV. BACKGROUND

[0005] An estimated 1.5 million new cases of human immunodeficiency virus (HIV) infection were diagnosed worldwide in 2021, and approximately 38.4 million people are currently living with AIDS / HIV. Although AIDS-related deaths have been dramatically reduced in recent years, an estimated 650,000 people nonetheless died from AIDS-related complications worldwide in 2021, and there remains no cure.Attorney Docket No.: HMJ-190-PCT

[0006] HIV is a retrovirus that infects CD4+ cells of the immune system, destroying or impairing their function. As the infection progresses, the immune system becomes weaker, leaving the infected person more susceptible to opportunistic infections and tumors, such as Kaposi’s sarcoma, cervical cancer, lymphoma, and neurological disorders. The most advanced stage of HIV infection is acquired immunodeficiency syndrome (AIDS). It can take 10-15 years for an HIV- infected person to develop AIDS, and certain antiretroviral drugs can delay the process even further.

[0007] With respect to HIV’s ability to enter into a cell, the envelope glycoprotein trimer (Env) is known to play a role in HIV virus attachment and subsequent entry into host cells. The mature Env trimer is comprised of non-covalently associated gp120-gp41 heterodimers that are formed by furin cleavage of a gp160 precursor. Kowalski et al., Science, 1987, 237:1351-1355.

[0008] The Env trimer is the only viral protein present on the surface of virions and HIV-1 infected cells; therefore, it represents a major antibody-targeted HIV-1 antigen. Env presentation to a host immune system elicits antibody responses against many diverse Env sites. These antibodies can impact HIV-1 through various mechanisms, including direct virus neutralization and Fc-effector activities, including antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) of infected cells.

[0009] The Env protein is metastable, heavily glycosylated, and highly diverse, due primarily to the acquisition of mutations. To counteract HIV-1 diversity, a vaccine should elicit broadly neutralizing antibodies (bnAbs) to cross-react with a variety of Env proteins. Structural biology studies have revealed the atomic details of the epitopes of numerous HIV-1 bnAbs, providing a potential roadmap for designing antigens that could integrate these bnAb epitopes. See, e.g., Wibmer et al., Curr. Opin. HIV AIDS, 2015, 10:135-143; Kwong et al., Immunity, 2018, 48:855- 871.

[0010] Although much effort has been put forth into designing effective therapeutics against HIV, currently no curative anti-retroviral drugs against HIV exist. While current antiretroviral (ART) therapies are able to control viral replication, they are unable to fully restore health or a normal immune status. ART-treated individuals still experience several co-morbidities, including increased cardiovascular disease, bone disorders and cognitive impairment. Additionally, therapy interruption leads to the re-emergence of viral replication and AIDS progression. Therefore, a need exists to develop improved therapeutics for HIV infection and AIDS.Attorney Docket No.: HMJ-190-PCT

[0011] The development of an effective vaccine against HIV remains a key challenge for fighting HIV infection. HIV-specific broadly neutralizing antibodies (bnAbs) could promote HIV remission by halting viral replication and clearing infected cells. However, there is a lack of vaccines that can induce bnAbs. Methods for effectively screening and identifying bnAbs are also needed. In addition, there is a need for reagents that match currently circulating HIV-1 sequences. SUMMARY

[0012] In one aspect, the present disclosure provides modified HIV-1 Envelope (Env) polypeptide and immunogenic fragments or variants thereof.

[0013] In some embodiments, the modified HIV-1 Env polypeptide comprises an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 116, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from N129D, T131S, D132N, G135D, G135E, G135D, S136L, S136Q, G137K, E143G, D175A, D175N, D267E, D267N, N268S, N268T A269F, A269G, N319D, N319T, R322D, R322Q, T323D, K324N, K324E, N326D, N326G, Q349K, V358I, M359I, Q375N, T380S, T380I, W381G, W381F, G385N, S386T, S390T, R438K, R438I, R438V, R438G, G440D, G440K, G441A, G441C, G441E, N442S, N443A, N443R, N443K, T446Q, T446K, E447G, T448N, G452Q, D455N, D455E, I496V, D655Q, G818T, G818L, L822I, L832S, and L837I; b) one or more repair modifications selected from S136T, S136N, E143D, A269T, N326E, T380S, G441A, G444N, T445S, T446N, and D655S; c) one or more trimer stabilization modifications selected from N290M, R292V, T306L, I516N, L537P, K569E, D570V, N632F, K636I, Q639V, A482C, T586C, and I540P; and / or d) a truncation after amino acid position 645 or amino acid position 693, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0014] In some embodiments, the modified HIV-1 Env polypeptide comprises an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 108, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from V84Q, N129K, N134R, M141I, K162D, N177K, K217R, E283K, Q303E, T304S, T304M, A307E, K329D, F349T, N350K, G354A, S363N, S392K, V414A, G443E, N444G, N445L, T448E, D457E, R540T, S619Q, D657N,Attorney Docket No.: HMJ-190-PCT A764S, I819M, C827Y, R831K, F841N, A844N, A844S, and Q846P; b) one or more repair modifications selected from K162Q, S392T T304A, and G443A; c) one or more trimer stabilization modifications selected from N292M, R294V, T308L, I518N, L539P, K571E, D572V, N634F, K638I, K641V, A484C, T588C, and I542P; and / or d) a truncation after amino acid position 647 or amino acid position 695, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0015] In some embodiments, the modified HIV-1 Env polypeptide comprises an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 105, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from H84N, N129S, N134K, G137N, N138T, S140E, G145S, E150K, K168P, N183S, K223S, N275G, N276S, A277V, G308S, E327D, N329I, T331N, K332Q, K332S, A341N, I354V, Q356T, P357L, M367I, K383R, N390S, G393K, S398G, G399N, G399E, G402R, V420D, R446A, G448S, G449E, G449N, N450S, N451G, N454K, N463F, T488S, R489S, R546K, E609R, N613G, N629D, N629S, D663E, R718S, G788E, L847I, and L852F; b) one or more repair modifications selected from S140T, E150D, K168Q, T331S, A341V, P357S, M367T, S398T, G449A, N451T, N463D, R489K, and E609D; c) one or more trimer stabilization modifications selected from N298M, R300V, T314L, I524N, L545P, K577E, D578V, N640F, K644I, K647V, A490C, T594C, and I548P; and / or d) a truncation after amino acid position 653 or amino acid position 701, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0016] In some embodiments, the modified HIV-1 Env polypeptide comprises an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 193, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from R320K, I365L, I366T, G372N, T381S, R460W, L457N, R460T, R460G, R460S, G462H, G463D, G463V, G463A, N464I, N464E, S466I, S466T, S467N, S467A, N469S, N469G, G475K, N478D, N478S, G644K, G644E, N678G, N678Y, R748Y, and N813D; b) one or more repair modifications selected from L81Q, Y92F, D129N, T163S, S168T, G172D, H184L, V330I, I434V, G463A, G475A, N478D, A538S, I587V, G623L, N678S, and N678T; c) one or more trimer stabilization modifications selected fromAttorney Docket No.: HMJ-190-PCT N309M, K311V, T327L, T539N, L560P, R592E, D593V, T655F, K659I, Q662V, A505C, P609C, and I563P; and / or d) a truncation after amino acid position 668 or amino acid position 716, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0017] In some embodiments, the modified HIV-1 Env polypeptide of the disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0018] In some embodiments, the modified HIV-1 Env polypeptide of the disclosure comprises the amino acid sequence of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ IDAttorney Docket No.: HMJ-190-PCT NO: 207, or an immunogenic fragment or variant thereof. In some embodiments, the modified HIV-1 Env polypeptide retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0019] In another aspect, provided herein is an artificial nucleic acid encoding any of the modified HIV-1 Env polypeptides disclosed herein. In some embodiments, the nucleic acid is a messenger RNA (mRNA) molecule. In some embodiments, the nucleic acid is a DNA molecule. Also provided are vectors comprising any of the artificial nucleic acids disclosed herein and host cells comprising such vectors.

[0020] In a further aspect, the disclosure provides a composition comprising any of the modified HIV-1 Env polypeptides disclosed herein, any of the artificial nucleic acids encoding such modified HIV-1 Env polypeptides, or any of the vectors comprising such artificial nucleic acids. In some embodiments, such compositions comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptidesAttorney Docket No.: HMJ-190-PCT selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or variant thereof. In some embodiments, such compositions comprise artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or variant thereof. In some embodiments, the compositions of the disclosure are immunogenic compositions.

[0021] In yet another aspect, provided herein is a vaccine comprising any of the compositions disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the disclosedAttorney Docket No.: HMJ-190-PCT vaccine further comprises an adjuvant. In some embodiments, the disclosed vaccine is an mRNA vaccine comprising at least one mRNA encoding the modified HIV-1 Env polypeptide described herein.

[0022] In a further aspect, the present disclosure provides a method of immunizing a subject, the method comprising administering to the subject in need thereof any of the vaccines disclosed herein. In some embodiments, the method disclosed herein prevents HIV-1 infection in the subject. In some embodiments, the method disclosed herein decreases the subject’s likelihood of getting HIV-1 infection. In some embodiments, the method disclosed herein reduces the subject’s likelihood of getting serious illness from HIV-1 infection. In some embodiments, the subject is a human. In some embodiments, the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A is an illustration of epitopes of broadly neutralizing antibodies (bnAb) and nearby hypervariable (HV) loops in the HIV Env protein. The illustration provides the location of HV loops and epitopes of representative HIV-1 bnAbs, including a V2HV loop around epitopes of V2-glycan antibodies VRC26.25 and PGT145, a V1HV loop around epitopes of V3-glycan antibodies 10-1074 and PGT135, and a V5HV loop around epitopes of CD4 binding site antibodies VRC01 and 3BNC117.

[0024] FIG. 1B is an illustration of epitopes of bnAbs and nearby HV loops in the HIV Env protein, illustrating the interference from long HV loops on the binding of potential broadly neutralizing antibodies targeting adjacent sites (left) and the improvement in accessibility generated after engineering shorter HV loops (right).

[0025] FIG. 2A is an illustration showing the impact of shorter loops on the antibody accessibility of epitope sites, wherein depth is an indicator of antibody accessibility of Env surface sites.

[0026] FIG. 2B is a plot showing a comparison of the depth of four representative antibodies (10-1074, PGT135, VRC01, and 3BNC117) before and after HV loop redesign in HIV B Env protein.

[0027] FIG. 2C is a plot showing a comparison of the depth of four representative antibodies (10-1074, PGT135, VRC01, and 3BNC117) before and after HV loop redesign in HIV C EnvAttorney Docket No.: HMJ-190-PCT protein.

[0028] FIG. 2D is a plot showing a comparison of the depth of four representative antibodies (10-1074, PGT135, VRC01, and 3BNC117) before and after HV loop redesign in HIV CRF01_AE Env protein.

[0029] FIG. 2E is an illustration showing a structural example of a redesigned HIV CRF01_AE Env protein to improve the accessibility of 10-1074 antibody epitope sites.

[0030] FIG. 3A is a plot showing a comparison of the bnAb PGT145 sensitivity (IC50µg / ml) for HIV-1 Env in both short and long V1HV, V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0031] FIG.3B is a plot showing a comparison of the bnAb VRC26.25 sensitivity (IC50µg / ml) for HIV-1 Env in both short and long V1HV, V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0032] FIG. 3C is a plot showing a comparison of the bnAb 10-1074 sensitivity (IC50 µg / ml) for HIV-1 Env in both short and long V1HV. V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0033] FIG. 3D is a plot showing a comparison of the bnAb PGT135 sensitivity (IC50 µg / ml) for HIV-1 Env in both short and long V1HV, V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0034] FIG. 3E is a plot showing a comparison of the bnAb VRC01 sensitivity (IC50 µg / ml) for HIV-1 Env in both short and long V1HV, V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0035] FIG.3F is a plot showing a comparison of the bnAb 3BNC117 sensitivity (IC50 µg / ml) for HIV-1 Env in both short and long V1HV, V2HV, V4HV, and V5HV loop regions, as described in Example 2.

[0036] FIG. 4A is an illustration showing clash sites between the bnAb 10-1074 and both a V1HV unmodified and a V1HV redesigned loop region for an Env sequence isolated from a person living with HIV, as described in Example 4.

[0037] FIG. 4B is an illustration showing clash sites between the bnAb PGT135 and both a V1HV unmodified and a V1HV redesigned loop region for an Env sequence isolated from a person living with HIV, as described in Example 4.

[0038] FIG. 4C is a plot showing a comparison of the depth (Å) of five Env variants from aAttorney Docket No.: HMJ-190-PCT person living with HIV, as described in Example 4, for the 10-1074 bnAb epitope sites, both before (left) and after (right) HV loop modification.

[0039] FIG. 4D is a plot showing a comparison of the depth (Å) of five Env variants from a person living with HIV, as described in Example 4, for the PGT135 bnAb epitope sites, both before (left) and after (right) HV loop modification.

[0040] FIG. 5A is a plot showing viral titers for Env consensus proteins with or without redesigned HV loops presented as mean + / - SEM of three replicates.

[0041] FIG. 5B is a plot showing negative-stain electron microscopy for stabilized trimers with and without redesigned loops for consensus subtypes B and C. Representative 2D class averages of putative monomeric or disassembled Env and intact trimers for stabilized trimers with and without redesigned HV loops corresponding to the consensus for subtypes B and C. Percentages above the images indicate the relative proportion of particles between monomer and trimer classes.

[0042] FIG. 5C is a plot showing 3D reconstructions of particles classified as trimers in 2D with no symmetry applied (C1). Classes resembling the closed, prefusion conformation of HIV-1 Env are shown in the left while classes resembling the open, prefusion conformation are shown in the right. Percentages above each map indicate the relative proportion between closed and open prefusion conformations. A side view for each reconstruction with the trimer apex on the bottom (top) and the trimer apex facing the viewer (bottom) is shown for each reconstruction.

[0043] FIG.6A-6J depict improved antibody binding to subtype B and CRF01_AE consensus Env with redesigned HV loops. FIG. 6A-6B depict comparison of the binding of plasma pools from 13 cohorts of people living with HIV-1 (FIG. 6A) and monoclonal antibodies to consensus B Env (FIG. 6B) with unmodified and redesigned HV loops. The number of plasma samples in each pool is shown in parenthesis in FIG. 6A. FIG. 6C-6D depict comparison of binding of plasma samples (FIG. 6C, n = 13) and monoclonal antibodies (FIG. 6D, n = 7, with PGDM1400 and MPER antibodies excluded) to the subtype B, C and CRF01_AE consensus Env with unmodified HV loops (left) and redesigned HV loops (right) using paired, non-parametric one- tailed Wilcoxon signed-rank tests. MPER antibodies and PGDM1400 were not included in the statistical tests as their epitopes are not presented on the gp140 glycoproteins. FIG. 6E-6J depict impact of HV loop modifications on the sensitivity of pseudoviruses to ten bnAbs. The neutralization sensitivity is reported as IC50values (μg / ml; FIG. 6E-6G) or IC80values (μg / ml;Attorney Docket No.: HMJ-190-PCT FIG. 6H-6J). Neutralization sensitivity (IC50or IC80values) is shown for the unmodified and redesigned HV loops and displayed separately for each Env clade (n = 10) (FIG. 6F, FIG. 6I) or each bnAb (FIG. 6G, FIG. 6J). The increase in sensitivity with the redesigned HV loops was tested for each clade with one-tailed exact Wilcoxon-Pratt signed-rank tests.

[0044] FIG. 7A-7B depict in vitro expression of HIV-1 Env proteins in mRNA-transfected mammalian cells as measured by flow cytometric surface staining by HIV-1 bNAbs, as described in Example 7. Median florescence intensity is reported for each naked multiple founder virus variant (MFV) mRNA construct grouped by HIV-1 subtype parent sequence. FIG. 7A: subtype B consensus (B.con) MFVs; FIG. 7B: subtype B (B036) MFVs. bNAbs used in surface staining are indicated along the x-axis. Horizontal bars indicate the median. A summary of the constructs used is provided in Table 9.

[0045] FIG. 8A depicts antigen-specific IgG end point titers at various times for mice immunized with LNPs encapsulating mRNA encoding subtype B consensus Env polypeptide and the indicated variants, as discussed in Example 8.

[0046] FIG. 8B depicts antigen-specific IgG end point titers at various times for mice immunized with LNPs encapsulating mRNA encoding subtype B B036 Env polypeptide and the indicated variants, as discussed in Example 8.

[0047] FIG. 9A depicts antigen-specific IgG end point titers at study week 8 (peak immunogenicity) for mice immunized with LNPs encapsulating mRNA encoding subtype B consensus Env polypeptide and the indicated variants, as discussed in Example 8.

[0048] FIG. 9B depicts antigen-specific IgG end point titers at study week 8 (peak immunogenicity) for mice immunized with LNPs encapsulating mRNA encoding subtype B B036 Env polypeptide and the indicated variants, as discussed in Example 8.

[0049] FIG. 10 depicts representative results obtained from the multiplex Luminex assay using the sera collected from immunized rabbits with eight gp140 Env antigens, as described in Example 8. The corresponding antigens are listed on the top of each graph. Gp140 ConB: Env gp140 consensus for subtype B; Gp140 ConAE: Env gp140 consensus for CRF01_AE; Gp140 ConA: Env gp140 consensus for subtype A; Gp140 ConC: Env gp140 consensus for subtype C; Gp140 ConD: Env gp140 consensus for subtype D; Gp140 ConG: Env gp140 consensus for subtype G; Gp140 ConAG: Env gp140 consensus for CRF02_AG; and Gp140 ConM: Env gp140 consensus for HIV-1 group M.Attorney Docket No.: HMJ-190-PCT

[0050] FIG.11 depicts a heatmap representing the neutralization titers measured against each pseudovirus (columns) in each immunized rabbit at each time point (rows), as described in Example 8. Data are shown for the 8 rabbits in a group at a specific visit. DETAILED DESCRIPTION

[0051] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the invention, and should not be interpreted as a limitation of the scope of the invention.

[0052] The present application discloses a set of novel recombinant immunogens for HIV generated by modifying the hypervariable loops of the consensus sequences of recently sampled HIV sequences in order to improve antibody-detection and / or for use as vaccine antigens. The immunogens disclosed herein surprisingly reduce or avoid hypervariable loop-mediated interference with anti-Env antibody binding by modifying the hypervariable loops and including less immunogenic sequences, while maintaining the structural integrity of the HIV Envelope (Env) protein. Definitions

[0053] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0054] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”

[0055] When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with theAttorney Docket No.: HMJ-190-PCT term “consisting of” or “consisting essentially of” to vary scopes of the disclosure.

[0056] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0057] As used herein, the term “HIV” refers to human immunodeficiency virus. HIV can be classified into two major subtypes (HIV-1 and HIV-2), each of which has many subtypes. In some embodiments, a human subject is infected with the HIV-1 subtype. In some embodiments, a human subject is infected with the HIV-2 subtype.

[0058] The term “antibody” refers to an immunoglobulin or antigen-binding fragment thereof, and encompasses any polypeptide comprising an antigen-binding fragment or an antigen-binding domain. The term includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. Unless preceded by the word “intact,” the term “antibody” includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Unless otherwise specified, an antibody is not necessarily from any particular source, nor is it produced by any particular method.

[0059] In certain embodiments, an antibody disclosed herein is a broadly neutralizing antibody (bnAb). In certain embodiments, an antibody disclosed herein is a non-neutralizing antibody. In certain embodiments, an antibody disclosed herein is a neutralizing antibody.

[0060] In certain embodiments, an antibody can be any of the five major classes of immunoglobulins, including IgA, IgD, IgE, IgG, and IgM, or subclasses thereof, based on the identity of their heavy-chain constant domains, which are referred to as alpha, delta, epsilon, gamma, and mu, respectively. In certain embodiments, the antibody is an IgG antibody.

[0061] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains and two identical heavy chains. In the case of an IgG antibody, the 4-chain unit is generally about 150,000 daltons. Each light chain is linked to a heavy chain by one covalentAttorney Docket No.: HMJ-190-PCT disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.

[0062] The term “neutralizing antibody” refers to an antibody that binds to a viral antigen and directly decreases or disrupts viral entry into a cell. A neutralizing antibody may inhibit the entry of HIV with a neutralization index of, for example, >1.5 or >2.0, discussed in Kostrikis et al., J. Virol., 1996, 70(1):445-458.

[0063] The term “broadly neutralizing antibody” or “bnAb” refers to an antibody that may bind to multiple versions of an antigen from different strains of a pathogen (e.g., virus), including for example multiple versions of a viral antigen (e.g., Env) from different HIV strains or isolates, each having unique genetic variations giving rise to different versions of the viral antigen (e.g., Env), and directly decrease or disrupt pathogen (e.g., HIV) entry into a cell. A bnAb may, in certain embodiments, neutralize multiple different genetic variants of a single virus (e.g., HIV).

[0064] The term “antigen” refers to a substance, such as a protein, a fragment thereof or a polysaccharide linked to a protein carrier, that when expressed in an animal or human cell or tissue is capable of triggering an immune response. The protein or fragment thereof may be glycosylated or non-glycosylated.

[0065] The term “epitope” refers to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0066] The term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen, immunogen, or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response. Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. An antibody response or humoral response is an immune response in which antibodies are produced. A “cellular immune response” is one mediated by T cells and / or other white blood cells.Attorney Docket No.: HMJ-190-PCT

[0067] The term “immunogen” or “immunogenic” refers to a compound, composition, or substance which is capable, under appropriate conditions, of stimulating an immune response, such as the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. As used herein, “immunize” means to induce in a subject a protective immune response against an infectious disease (e.g., HIV).

[0068] As used herein, an “artificial nucleic acid molecule” or the like may typically be understood to be a nucleic acid, e.g., a DNA or an RNA, that does not occur naturally. Thus, an “artificial messenger ribonucleic acid (mRNA)” refers to an mRNA that does not occur naturally. In other words, an artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule or mRNA molecule. Such nucleic acid or mRNA molecule may be non-natural due to its individual sequence (which does not occur naturally) and / or due to other modifications, e.g., structural modifications of nucleotides that do not occur naturally. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule (e.g., mRNA), or a hybrid molecule comprising DNA and RNA portions. Typically, artificial nucleic acid molecules may be designed and / or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). Further, the term “artificial nucleic acid molecule” or the like (e.g., “artificial mRNA”) is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

[0069] The term “in some embodiments” or “in certain embodiments” or the like refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0070] The term “sequence identity” refers to the similarity between amino acid or nucleic acid sequences expressed in terms of the similarity between the sequences. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods.

[0071] The terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimalAttorney Docket No.: HMJ-190-PCT alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison,” in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0072] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0073] In some embodiments, the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides. In some embodiments, the degree of identity is given for the entire length of the reference sequence.

[0074] Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional and / or structural property of said given sequence, e.g., and in some instances, are functionally and / or structurally equivalent to said given sequence. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally and / or structurally equivalent to said givenAttorney Docket No.: HMJ-190-PCT sequence.

[0075] As used here, an “mRNA vaccine” refers to a type of vaccine that uses messenger RNA (mRNA) to induce an immune response. In some embodiments, the mRNA is encapsulated in a lipid nanoparticle.

[0076] The term “subject” refers to any animal, such as a mammal, including humans, non- human primates, rodents, and the like which is to be the recipient of a particular treatment.

[0077] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring.

[0078] The term “preventing” when used in the context of a disease or disease condition means prophylactic administration of a composition that stops or otherwise delays the onset of a pathological hallmark or symptom of a disease or disorder.

[0079] The term “treating” when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a molecular level, the biochemical basis of the disease, such as halting replication of a virus, etc.

[0080] The term “vaccine composition” or “vaccine” refers to a composition that generates a protective immune response in a subject. As used herein, a “protective immune response” refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection) or reduces the symptoms of infection (for instance an infection by HIV). Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response.

[0081] The term “vaccinate” or the like refers to the administration of a vaccine composition to generate a protective immune response in a subject, for example to a disease-causing agent suchAttorney Docket No.: HMJ-190-PCT as a virus, e.g., HIV. Vaccination can occur before, during, and / or after exposure to a disease- causing agent, and / or to the development of one or more symptoms, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. Modified Hypervariable Loop Regions of HIV Env Protein Epitopes

[0082] To be effective against the various highly-diverse HIV-1 strains circulating globally, an HIV-1 vaccine should be capable of eliciting bnAbs against Env. Disclosed herein are novel Env variable loop designs that mediate bnAb accessibility to Env, in particular hypervariable (HV) loops designs, i.e., the hypervariable segment that is present in the variable loops 1, 2, 4 and 5 of Env.

[0083] Known HIV-1 bnAbs cover the Env surface and are grouped in categories based on the location of their epitopes. Three epitope specificities were considered herein that are preferentially targeted by bnAbs from people living with HIV-1: (1) the Env variable loop 2 (V2)-apex epitope, (2) the glycan supersite; and (3) the CD4 binding site (CD4bs). These three epitope sites of bnAbs may be under certain functional constraints. For example, the V2-apex epitope shields V3 and stabilizes the prefusion Env trimer. The glycan-supersite epitope allows the release of V3 from the prefusion conformation to reach the co-receptors after receptor binding, and the CD4bs epitope is involved in receptor binding.

[0084] HIV-1 Env presents five variable loops and four of them (V1, V2, V4, V5) contain a hypervariable segment, called the hypervariable (HV) loop. These HV loops can be adjacent to epitope sites. The V1 HV loop (V1HV) is near the glycan-supersite epitope and is targeted by the antibodies 10-1074 and PGT135. Gristick et al., Nat. Struct. Mol. Biol., 2016, 23:906-915; Kong et al., Nat. Struct. Mol. Biol., 2013, 20:796-803.

[0085] The V2 HV loop (V2HV) is near the V2-apex epitope and is targeted by the antibodies PGT145 and VRC26.25. Lee et al., Immunity, 2017, 46:690-702; Liu et al., Nat. Struct. Mol. Biol., 2017, 24:370-378; Gorman et al., Cell Rep., 2020, 31:107488.

[0086] The V5 HV loop (V5HV) is near the CD4bs epitope and is targeted by the antibodies VRC01 and 3BNC117. Zhou et al., Science, 2010, 329:811-817; Klein et al., Cell, 2013, 153:126- 138.

[0087] These HV loops can occlude access to the adjacent bnAb epitopes and are typically targeted by antibodies to elicit strain-specific responses rather than the more functional bnAbAttorney Docket No.: HMJ-190-PCT specificities they are shielding. Notably, these three antibody specificities accounted for more than half of the neutralizing antibodies in two natural infection cohorts. In the IAVI Protocol C, neutralization breadth was tested in 2220 samples collected from 439 people living with HIV in East Africa. Broad neutralizers were determined by a neutralization score ≥ 1, which approximately predicted a breadth ≥50% on a 105-virus panel; the three specificities corresponded to 57% of dominant specificities of top 42 neutralizers. Landais et al., PLoS Pathog., 2016, 12:e1005369. In the RV217 cohort, neutralizing responses were measured two to four years after diagnosis for 70 participants against a panel of 34 viruses and breadth was defined as recognition of 70% of strains. The three specificities account for 65% (33 / 51) of delineated epitope specificities. Townsley et al., Cell Host Microbe, 2021, 29:564-578.e9.

[0088] This application demonstrates that modifying Env hypervariable loops provides enhanced access to bnAb epitopes on Env. As disclosed herein, Env sequences with the longest cognate hypervariable loops showed decreased sensitivity to bnAbs when compared to Env with the shortest hypervariable loops (p≤0.015). Since centralized antigens capture optimal Env features, hypervariable loops were redesigned for updated HIV-1 Env consensus sequences based on sequences sampled since 2010 from subtypes B and C and a circulating recombinant form 01_AE (CRF01_AE). V1HV, V2HV, and V5HV were redesigned to be shorter while maintaining the integrity of the Env structure and glycan shield, and V4HV was modified while maintaining the integrity of the Env structure and glycan shield. Typically, the hypervariable loop was shortened by at least 3 amino acids relative to the consensus hypervariable loop sequences. In some embodiments, one or more hypervariable loops in a modified HIV-1 Env polypeptide is shortened by 1-10, 2-8, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 amino acids as compared to a wild type or consensus HIV-1 Env sequence. In certain embodiments, each hypervariable loop in the modified HIV-1 Env polypeptide or immunogenic fragment or variant thereof is shortened by 1-10, 2-8, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, or 3-9 amino acids as compared to a wild type or consensus HIV-1 Env sequence.

[0089] The redesigned variable loop structures improved the antibody accessibility to the bnAbs, i.e., the closest distance between an epitope site and the antibody probe diminished, especially for the glycan-supersite epitope. Additionally, the redesigned hypervariable loops were modified to contain mainly glycine and serine that lack antibody-recognizable side chains, thus minimizing the strain-specific targeting typically associated with the Env variable loops.Attorney Docket No.: HMJ-190-PCT Accordingly, in certain embodiments, the modified HIV-1 Env polypeptide comprises one or more amino acid substitutions in one or more of the hypervariable loops. In certain embodiments, one or more amino acids in a hypervariable loop of the modified HIV-1 Env polypeptide is substituted for a glycine and / or a serine. In certain embodiments, one or more amino acids in at least 2, 3, or 4 hypervariable loops of the modified HIV-1 Env polypeptide are substituted for a glycine and / or a serine. In certain embodiments, at least 1-10, such as 2-8, amino acids in the hypervariable loops of the modified HIV-1 Env polypeptide are substituted for a glycine and / or a serine. In other embodiments, at least 2, 4, 6, 8, or 10 amino acids in a hypervariable loop of the modified HIV-1 Env polypeptide are substituted for a glycine and / or a serine. Table 1 below lists the sequences for each of the redesigned Env hypervariable loops for V1HV, V2HV, V4HV, and V5HV, including subtypes A1, B, C, D, G, CRF01_AE, CRF02_AG, and M. Table 1. Redesigned Env Hypervariable Loops. Hyper Variable Subtype Original sequences Redesigned O: O: 9) 0) : O: Q )Attorney Docket No.: HMJ-190-PCT V1 CRF02_ SNANNSTSNNRNITNEMKG SNVNGSGGNMTG (SEQ ID NO: AG (SEQ ID NO: 7) 45) : : O: O: D O: O: D 2) O:Attorney Docket No.: HMJ-190-PCT WNGNGNTTEGCNGT (SEQ ID WNGNGSGNGSGSNGT (SEQ ID NO: 144) NO: 75) O: D O: D

[0090] For each modified loop, the anchor sites, which were relatively more conserved and located at the C / N-terminal of the hypervariable loops, were fixed. Those sites interacted with the non-hypervariable part of Env and anchored the hypervariable loops. Spacers were then iteratively tested to identify a spacer with a length that was compatible with the conformation of the rest of Env, as well as with potential N-linked glycosylation sites to keep the glycan shield of Env intact. The design iteration also sought to make C / N-anchoring sites more representative of circulating viruses.

[0091] In addition to reducing HV loop length, in certain embodiments disclosed herein, the immunogenicity of the HV loop is reduced as compared to the unmodified loop by using shorter spacers composed of less immunogenic amino acids. In certain embodiments, the redesigned spacers only contain glycine and serine except for the potential N-linked glycosylation siteAttorney Docket No.: HMJ-190-PCT (PNGS). This is unlike natural HV loops, which contain various amino acids. Since glycine and serine lack antibody-recognizable side chains, the redesigned loops may in certain embodiments be less likely to act as decoys attracting strain-specific antibody responses. A recent study found that long V1V2s in CRF01_AE Env were associated with the more rapid development of autologous neutralization in participants from the RV217 cohort. Kuriakose et al., J. Virol., 2023, e0163522. The results highlight the potential role of long HV loops in the development of strain- specific responses, as strain-specific responses could be considered undesirable because autologous neutralization was not associated with the development of neutralization breadth in this cohort. Modified Consensus Sequences for HIV-1 Subtypes Based on Redesigned Hypervariable Loops

[0092] Consensus sequences for sequences sampled after 2010 for each of the HIV-1 subtypes A1 (n=53), B (n=462), C (n=171), and for CRF01_AE (n=134) were generated, and consensus sequences for sequences sampled after 2000 for each of subtypes D (n=63), G (n=62), and CFR02_AG (n=131) were generated. Each consensus was determined by the most common residue at each site, and ties were broken in the order of L, G, I, K, E, A, V, T, R, Q, S, P, N, D, W, Y, F, H, C, and then M. Sites with greater than 50% gaps in the subtype alignment were excluded. The group M consensus was a consensus of the consensus sequences for the different subtypes and CRFs; this was done without weighting by subtype / CRF prevalence. Gaps in the group M consensus, where no residue was dominant among consensus sequences for different subtypes and CRFs, were replaced with the subtype C residue.

[0093] Based on the modeled structures of each consensus using AlphaFold2 with ColabFold, the hypervariable loops were redesigned to make them short yet still compatible with structural constraints. In some embodiments, SEQ ID NO: 96-119 as disclosed herein or immunogenic fragments or variants thereof are modified consensus sequences that reflect current HIV-1 diversity for subtypes A1, B, C, D, and G, and CRF01_AE, CRF02_AG, as well as group M.

[0094] In some embodiments, the modification of potential N-glycan sites (PNGS) retains the glycan shield, i.e., an array of N-linked glycans on the HIV-1 envelope glycoprotein, which can contribute to immunogenicity. In certain embodiments, the modified consensus sequence, such as the modified consensus sequence of an HIV-1 Env subtype B (e.g., SEQ ID NO: 97 or 116) comprises a variable loop 3 (V3HV) of CTRPNNNTRKSIHIGPGRAFYATGDIIGDIRQAHC (SEQ ID NO: 120). In some embodiments, the hypervariable loops of the consensus sequencesAttorney Docket No.: HMJ-190-PCT provided herein include a GS-linker. In some embodiments, the S in the GS-linker is replaced to form “NxS,” wherein x indicates any amino acid with the exception of proline, e.g., such that the NxS may be a PNGS site. The modified consensus sequence for each of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, and SEQ ID NO: 118 is set forth below in Table 2. Also shown in Table 2 are the original (unmodified) consensus sequences for each of SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, representing each of HIV-1 Env subtypes CRF01_AE, D, C, CRF02_AG, A, G, B, and M, respectively. Table 2. Modified and Original Consensus Sequences with Shortened Hypervariable Loops. HIV- Modified Modified and Original Consensus Sequence 1 or N S F D I I R A G N E T : P L V FAttorney Docket No.: HMJ-190-PCT TDNAKTIIVQLNESVEINCTRPNNNTRKSIHIGPGRAFYATGDIIGD IRQAHCNISRTKWNNTLKQIVKKLREQFGNKTIIFNQSSGGDPEIV II A L G I E R A P D F A L V A D I I R I RAttorney Docket No.: HMJ-190-PCT KHICTTNVPWNSSWSNKSLDEIWNNMTWMEWEREIDNYTGLIYS LIEESQIQQEKNEQELLELDKWASLWNWFSITNWLWYIKIFIMIV I G V N I I I I S V N F I I T G P S E N G I I I A L QAttorney Docket No.: HMJ-190-PCT CRF0 Modified MRVMGTQRNYPLLWRWGMIIFWIIIICNAENLWVTVYYGVPVW 2_AG RDAETTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIHLENV E D I I R A G N E N E Q F R S Y R T V N V NAttorney Docket No.: HMJ-190-PCT GSLAEEEIIIRSENLTNNAKTIIVHLNKSVEINCTRPSNNTRTSIRIGP GQVFYRTGDIIGDIRKAYCEINGTKWNETLKQVAEKLKEHFNNK E Q D H S V N I I T G I E Q P I I P F Q S N G AAttorney Docket No.: HMJ-190-PCT IEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGK HICTTNVPWNSSWSNKSLDEIWNNMTWMEWEREIDNYTGLIYSL G P P L F A T L P K R G P G E I Y Y I IAttorney Docket No.: HMJ-190-PCT ID NO: 108) MRVRGILRNYQQWWIWGILGFWMLMICNVVGNLWVTVYYGVP V K I I P G L L N G R V V N G I Y I F VAttorney Docket No.: HMJ-190-PCT YRLDVVQIDESSNSSQYRLINCNTSAITQACPKVSFEPIPIHYCAPA GFAILKCNDKEFNGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLA Q I L V F P N S N S D P G Y R I N S S N SAttorney Docket No.: HMJ-190-PCT GLILTRDGGNNNNTNETFRPGGGDMRDNWRSELYKYKVVKIEPL GVAPTRAKRRVVEREKRAVGLGAVFLGFLGAAGSTMGAASITLT A S R L I V N I T P G L G T V N P L A T T G A LAttorney Docket No.: HMJ-190-PCT QREPDRPERIEEGGGEQDRDRSIRLVSGFLALAWDDLRSLCLFSY HRLRDFVLIAARTVELLGRSSLQGLRLGWEGLKYLWNLLLYWG P L P T I V K T K C L I I A P L A A S P T N G L T A L L SAttorney Docket No.: HMJ-190-PCT WKDAETTLFCASDAKAYETEVHNVWATHACVPTDPNPQEIVLE NVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLN V N E I E S G I I I A A S TI V Q N L RCombinations of Redesigned HV Loops and Other Types of Modifications

[0095] The redesigned HV loops disclosed herein can be combined with other strategies to further improve the accessibility of bnAb epitopes. For instance, without wishing to be bound by any theory, while bnAb epitopes near HV-loops can be made more accessible after HV loopAttorney Docket No.: HMJ-190-PCT redesign, their accessibility may still be outcompeted by targeting of strain-specific glycan holes or by targeting of the base of the trimer, which is common when a soluble, stabilized prefusion Env is administered. Thus, fixing strain-specific glycan holes, shielding the Env trimer base (e.g., by mounting it on a liposome or co-delivering it with Gag in an mRNA vaccine) may further improve the accessibility of bnAb epitopes. Shao et al., Nat. Chem., 2015, 7:438-446; Zhang et al., Nat. Med., 2021, 27:2234-2245. Moreover, the epitope near the redesigned HV-loops can also be optimized, e.g., if mutations are found in key epitope sites, the mutated residue(s) may be reverted back to the consensus amino acid(s) at that site. Furthermore, many unshielded epitopes are exposed on the post-fusion Env. Thus, a stabilized prefusion Env trimer can also improve functionality, as the improved bnAb accessibility may be insignificant in the post-fusion context. This can be achieved with, for example, the repair and stabilization procedure which systematically fixes rare mutations and stabilizes HIV-1 Env in the prefusion conformation. Rutten et al., Cell Reports, 2018, 23:584-595; Rawi et al., Cell Reports, 2020, 33:108432. Additionally, it has reported that infections established with multiple founder variants are associated with the development of neutralization breadth years later. Lewitus et al., PLoS Pathogens., 2022, 18(3):e1010369. Thus, design strategy that integrates the variability observed in acute HIV-1 infection with multiple founder variants can also be used to further improve the accessibility of bnAb epitopes. In addition to the aforementioned additional modifications to improve the accessibility of bnAb epitopes, the modified consensus sequences for HIV-1 subtypes disclosed herein can also be modified to increase their solubility or make them membrane-bound depending on the vaccine platforms used.

[0096] Accordingly, in certain embodiments, one or more of the following additional modifications can be introduced into the modified consensus sequences for HIV-1 subtypes based on redesigned HV loops disclosed herein: (1) multiple founder variant modifications, (2) repair modifications and trimer stabilization modifications, and (3) solubility / membrane-bound modifications, as described in more detail below. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more multiple founder variant modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more repair modifications and one or more trimer stabilization modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypesAttorney Docket No.: HMJ-190-PCT disclosed herein further comprise one or more solubility / membrane-bound modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more multiple founder variant modifications, one or more repair modifications, and one or more trimer stabilization modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more multiple founder variant modifications and one or more solubility / membrane-bound modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more repair modifications, one or more trimer stabilization modifications, and one or more solubility / membrane-bound modifications. In certain embodiments, the modified consensus sequences with HV loops modifications for HIV-1 subtypes disclosed herein further comprise one or more multiple founder variant modifications, one or more repair modifications, one or more trimer stabilization modifications, and one or more solubility / membrane-bound modifications. Multiple Founder Variant Modifications

[0097] Optimal sequence-based design for multi-antigen HIV-1 vaccines using minimally distant antigens has been previously reported. Lewitus et al., PLoS Comput. Biol., 2022, 18(10):e1010624. Briefly, by training an HIV-1 Env consensus sequence on the diversity from acute infections with multiple founders, a set of minimally distant antigens that is representative of the diversity seen in a multi-founder acute infection can be derived in silico. The probabilistic model to simulate Env sequences that replicated the variability of infections with multiple founder variants disclosed in Lewitus et al., supra (incorporated herein by reference), can be used to produce multiple founder variant modifications specific to any given HIV-1 subtype or circulating recombinant form (CRF).

[0098] Accordingly, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype B of SEQ ID NO: 116 can include one or more of the following multiple founder variant modifications: N129D, T131S, D132N, G135D, G135E, G135D, S136L, S136Q, G137K, E143G, D175A, D175N, D267E, D267N, N268S, N268T A269F, A269G, N319D, N319T, R322D, R322Q, T323D, K324N, K324E, N326D, N326G, Q349K, V358I, M359I, Q375N, T380S, T380I, W381G, W381F, G385N, S386T, S390T, R438K, R438I, R438V, R438G, G440D, G440K, G441A, G441C, G441E, N442S, N443A, N443R, N443K, T446Q, T446K, E447G, T448N, G452Q, D455N, D455E, I496V, D655Q, G818T, G818L, L822I, L832S,Attorney Docket No.: HMJ-190-PCT and L837I.

[0099] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype C of SEQ ID NO: 108 can include one or more of the following multiple founder variant modifications: V84Q, N129K, N134R, M141I, K162D, N177K, K217R, E283K, Q303E, T304S, T304M, A307E, K329D, F349T, N350K, G354A, S363N, S392K, V414A, G443E, N444G, N445L, T448E, D457E, R540T, S619Q, D657N, A764S, I819M, C827Y, R831K, F841N, A844N, A844S, and Q846P.

[0100] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 CRF01_AE of SEQ ID NO: 105 can include one or more of the following multiple founder variant modifications: H84N, N129S, N134K, G137N, N138T, S140E, G145S, E150K, K168P, N183S, K223S, N275G, N276S, A277V, G308S, E327D, N329I, T331N, K332Q, K332S, A341N, I354V, Q356T, P357L, M367I, K383R, N390S, G393K, S398G, G399N, G399E, G402R, V420D, R446A, G448S, G449E, G449N, N450S, N451G, N454K, N463F, T488S, R489S, R546K, E609R, N613G, N629D, N629S, D663E, R718S, G788E, L847I, and L852F. Repair Modifications and Trimer Stabilization Modifications

[0101] A universal approach to optimize the folding and stability of prefusion-closed HIV-1 Env trimers has previously been described in Rutten et al., Cell Reports, 2018, 23:584-595. Briefly, the approach contains a repair stage that utilizes a consensus strategy to substitute rare strain-specific residues with more prevalent ones and a stabilization stage that involves structure- based design and experimental assessment confirmed by crystallographic feedback. Based on this concept, an automated procedure to produce soluble, prefusion-closed Env trimers from a majority of HIV-1 strains was described in Rawi et al., Cell Reports, 2020, 33:108432. Any of the methods disclosed in Rutten et al. and Rawi et al., both incorporated herein by reference, can be used to produce repair modifications and trimer stabilization modifications specific to any given HIV-1 subtype or circulating recombinant form (CRF).

[0102] Accordingly, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype B of SEQ ID NO: 116 can include one or more of the following repair modifications: S136T, S136N, E143D, A269T, N326E, T380S, G441A, G444N, T445S, T446N, and D655S, and one or more of the following trimer stabilization modifications: N290M, R292V, T306L, I516N, L537P, K569E, D570V, N632F, K636I, Q639V, A482C, T586C, and I540P.Attorney Docket No.: HMJ-190-PCT

[0103] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype C of SEQ ID NO: 108 can include one or more of the following repair modifications: K162Q, S392T T304A, and G443A, and one or more of the following trimer stabilization modifications: N292M, R294V, T308L, I518N, L539P, K571E, D572V, N634F, K638I, K641V, A484C, T588C, and I542P.

[0104] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 CRF01_AE of SEQ ID NO: 105 can include one or more of the following repair modifications: S140T, E150D, K168Q, T331S, A341V, P357S, M367T, S398T, G449A, N451T, N463D, R489K, and E609D, and one or more of the following trimer stabilization modifications: N298M, R300V, T314L, I524N, L545P, K577E, D578V, N640F, K644I, K647V, A490C, T594C, and I548P. Solubility / Membrane-Bound Modifications

[0105] The HIV-1 Env consists of two subunits, the surface glycoprotein (gp120) at the N- terminus and the transmembrane glycoprotein (gp41) at the C-terminus. The transmembrane glycoprotein includes a fusion domain, also known as fusion peptide, which consists of a hydrophobic stretch of about 20 amino acid residues at the N‐terminus, two heptad repeats HR1 and HR2, transmembrane domain, three stretches of residues between these four major regions, and a cytoplasmic tail. To increase the solubility, the majority of the transmembrane glycoprotein can be removed from the modified consensus sequences for HIV-1 subtypes disclosed herein. On the other hand, keeping the transmembrane glycoprotein, or at least part thereof containing the transmembrane domain, can make the modified consensus sequences for HIV-1 subtypes disclosed herein remain membrane-bound. For example, modifications can be made by removing the cytoplasmic tail from the modified consensus sequences for HIV-1 subtypes disclosed herein while maintaining the resultant polypeptide membrane-bound.

[0106] Accordingly, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype B of SEQ ID NO: 116 can be truncated after amino acid position 645 to make the protein more soluble. Alternatively, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype B of SEQ ID NO: 116 can be truncated after amino acid position 693 to keep the protein membrane-bound while removing the cytoplasmic tail.

[0107] In certain embodiments, the modified consensus sequence with HV loopsAttorney Docket No.: HMJ-190-PCT modifications for HIV-1 subtype C of SEQ ID NO: 108 can be truncated after position amino acid 647 to make the protein more soluble. Alternatively, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype C of SEQ ID NO: 108 can be truncated after amino acid position 695 to keep the protein membrane-bound while removing the cytoplasmic tail.

[0108] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 CRF01_AE of SEQ ID NO: 105 can be truncated after amino acid position 653 to make the protein more soluble. Alternatively, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 CRF01_AE of SEQ ID NO: 105 can be truncated after amino acid position 701 to keep the protein membrane-bound while removing the cytoplasmic tail. Exemplary Modified Consensus Sequences for HIV-1 Subtypes Comprising Redesigned HV Loops and Other Types of Modifications

[0109] Exemplary modified consensus sequences for HIV-1 subtypes comprising one or more redesigned HV loops disclosed herein and one or more aforementioned modifications are provided in Tables 3A-3C below. Table 3A. Exemplary Modified Consensus Sequences for HIV-1 Subtype B Comprising Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 116 (“B_2010s_hv_redesigned.O1.Seed”) Name Sequence B 2 1 h i MRVK IRKNY HLWRW TMLL MLMI AAE LWVTVYY V V V E I D P G V G EAttorney Docket No.: HMJ-190-PCT B_2010s_hv_redesi MRVKGIRKNYQHLWRWGTMLLGMLMICSAAEQLWVTVYYGV gned.O1.Seed.RnS. PVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEV V V E I D P G V W V V S I V Q L D G L L V V V E I D CAttorney Docket No.: HMJ-190-PCT RIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTKDGGSNGS QENFRPGGGNMRDNWRSELYKYKVVKIEPLGVAPTKCKRRVV G E V V V E I D C S W V V S D P G V Q L D G L L V VAttorney Docket No.: HMJ-190-PCT TLNCTNLNENGTMEKGDIKNCSFNITTSIRDKVQKEYALFYKLD VVPIANTSYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCN S D P G V G E V V S D P G V W V V V E II P L I R RAttorney Docket No.: HMJ-190-PCT LRDLLLIVARIVELLGRRGWEALKYWWNLLQYWSQELKNSAVS LLNATAIAVAEGTDRIIEVVQRALRAILHIPRRIRQGLERALL V V V E I D P N V V V E I D P N R W V V V E II P G VAttorney Docket No.: HMJ-190-PCT QREKRAVGIGAMFLGFLGAAGSTMGAASITLTVQARQLLSGIVQ QQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLL D G L L V V V E I D P G V G E V V V E I D P G V WTable 3B. Exemplary Modified Consensus Sequences for HIV-1 Subtype C Comprising Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 108 (“C_2010s_hv_redesigned.O1.Seed”).Attorney Docket No.: HMJ-190-PCT Name Sequence C_2010s_hv_redesi MRVRGILRNYQQWWIWGILGFWMLMICNVVGNLWVTVYYGV M V K L II L G N V V K L II L G N A R V L R R G G L LAttorney Docket No.: HMJ-190-PCT LGIWGCSGKLICTTAVPWNSSWSNKSQEEIWDNMTWMQWDREI SNYTDTIYRLLEESQNQQEKNEKDLLALDSWNNLWNWFDITNW R L V L R R G G N Q D V L R R G G N R Y M F V K L IIAttorney Docket No.: HMJ-190-PCT GDIIGDIRQAHCNISESKWNKTLQRVSKKLKEHFPNKTIKFKPSSG GDLEITTHSFNCRGEFFYCNTSKLFNGTYNGNGSGNGSGSNSTIT N I N P H S V K L II L G N V V K L II L G N A RAttorney Docket No.: HMJ-190-PCT C_2010s_hv_redesi MRVRGILRNYQQWWIWGILGFWMLMICNVVGNLWVTVYYGV gned.O1.t820A PVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEM V K L II I R I N P H V K L II L G T N V ) V K L II L G T N VAttorney Docket No.: HMJ-190-PCT VEGGGGSGGGGSAVGIGAVFLGFLGAAGSTMGAASNTLTVQAR QLLSGIVQQQSNLPRAPEAQQHMLQLTVWGIKQLQARVLAIERY M V K L II T A N I N P H V K L II L G N V V K LAttorney Docket No.: HMJ-190-PCT KCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIII RSENLTNNAKTIIVHLNESVEIVCTRPNNMTVKSIRIGPGQAFYAL G N A R T. p y q _ p ng Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 105 (“01_AE_2010s_hv_redesigned.O1.Seed”). Name Sequence 01 AE 2010s hv MRVKETQMNWPNLWKWGTLILGLVIICSASDNLWVTVYYGVP L A V G C Y L A V G AAttorney Docket No.: HMJ-190-PCT KRRVVEGGGGSGGGGSAVGIGAMIFGFLGAAGSTMGAASNTLT VQARQLLSGIVQQQSNLPRAPEAQQHLLQLTVWGIKQLQARVL L L A V F G A L D H G L A V F G C Y L AAttorney Docket No.: HMJ-190-PCT LFYKLDIVPIGNSSEYRLINCNTSVIKQACPKISFDPIPIHYCTPAG YAILKCNDKNFNGTGPCKNVSSVQCTHGIKPVVSTQLLLNGSLA V F G A T L L Y E P K I R E E L A VAttorney Docket No.: HMJ-190-PCT YLEVQKFLGLWGCSGKIICCTAVPWNSTWSNKSYEEIWGNMTW IEWEREISNYTNQIYEILTESQFQQDINEVDLLELD (SEQ ID NO: L A V T L L A G A L D H G L AAttorney Docket No.: HMJ-190-PCT EEEIIIRSENLTNNAKTIIVHLNKSVEINCTRPSNMTVTSIRIGPGQV FYRLGDIIGDIRKAYCEINGTKWNETLKQVAEKLKEHFNNKTIIF G C Y L A V G A T L L V T S S E H GAttorney Docket No.: HMJ-190-PCT QELKTSAISLLDATAIAVAGWTDRVIEVAQRAWRAILHIPRRIRQ GLERALL (SEQ ID NO: 190) L V T S K L ) L V T S K W

[0110] The modifications made to the exemplary modified consensus sequences for HIV-1 subtypes comprising redesigned HV loops and other types of modifications provided in Tables 3A-3C are summarized in Tables 4A-4C. Table 4A. Summary of Modifications Made to the Exemplary Modified Consensus Sequences for HIV-1 Subtype B Comprising Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 116 Provided in Table 3AAttorney Docket No.: HMJ-190-PCT (Amino Acid Position Is Indexed by Referenced to SEQ ID NO: 116). Name SEQ MFV Repair Trimer Furin Truncation ID Modifications Modifications Stabilization Cleavage afterAttorney Docket No.: HMJ-190-PCT R438K, N442S, A482C, T446Q, T448N, T586C, I540PAttorney Docket No.: HMJ-190-PCT S390T, N443K, N632F, K636I, G452Q I540PAttorney Docket No.: HMJ-190-PCT membra N632F, K636I, ne I540PHIV-1 Subtype C Comprising Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 108 Provided in Table 3B (Amino Acid Position Is Indexed by Referenced to SEQ ID NO: 108). Name SEQ MFV Repair Stabilization Furin Truncation ID Modifications Modifications Modifications Cleavage afterAttorney Docket No.: HMJ-190-PCT C_2010 169 V84Q, M141I, K162Q, S392T N292M, 491-494: 695 s_hv_re K217R, E283K, R294V, REKR-Attorney Docket No.: HMJ-190-PCT A484C, T588C, I542PTable 4C. Summary of Modifications Made to the Exemplary Modified Consensus Sequences for HIV-1 CRF01_AE Comprising Redesigned HV Loops and Other Types of Modifications Derived from the Modified Consensus Sequence of SEQ ID NO: 105 Provided in Table 3C (Amino Acid Position Is Indexed by Referenced to SEQ ID NO: 105). Name SEQ Multiple Repair Stabilization Furin TruncationAttorney Docket No.: HMJ-190-PCT v_redesi T314L, I524N, >RRRRR gned.O1 L545P, RAttorney Docket No.: HMJ-190-PCT K332Q, A341N, P357L,Attorney Docket No.: HMJ-190-PCT A490C, T594C, I548P

[0111] Accordingly, in certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype B of SEQ ID NO: 116, or a variant thereof having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 116, includes one or more multiple founder variant modifications selected from N129D, T131S, D132N, G135D, G135E, G135D, S136L, S136Q, G137K, E143G, D175A, D175N, D267E, D267N, N268S, N268T A269F, A269G, N319D, N319T, R322D, R322Q, T323D, K324N, K324E, N326D, N326G, Q349K, V358I, M359I, Q375N, T380S, T380I,Attorney Docket No.: HMJ-190-PCT W381G, W381F, G385N, S386T, S390T, R438K, R438I, R438V, R438G, G440D, G440K, G441A, G441C, G441E, N442S, N443A, N443R, N443K, T446Q, T446K, E447G, T448N, G452Q, D455N, D455E, I496V, D655Q, G818T, G818L, L822I, L832S, and L837I, one or more repair modifications selected from S136T, S136N, E143D, A269T, N326E, T380S, G441A, G444N, T445S, T446N, and D655S, one or more trimer stabilization modifications selected from N290M, R292V, T306L, I516N, L537P, K569E, D570V, N632F, K636I, Q639V, A482C, T586C, and I540P, and / or a truncation after amino acid position 645 or amino acid position 693. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0112] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 subtype C of SEQ ID NO: 108, or a variant thereof having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 108, includes one or more multiple founder variant modifications selected from V84Q, N129K, N134R, M141I, K162D, N177K, K217R, E283K, Q303E, T304S, T304M, A307E, K329D, F349T, N350K, G354A, S363N, S392K, V414A, G443E, N444G, N445L, T448E, D457E, R540T, S619Q, D657N, A764S, I819M, C827Y, R831K, F841N, A844N, A844S, and Q846P, one or more repair modifications selected from K162Q, S392T T304A, and G443A, one or more trimer stabilization modifications selected from N292M, R294V, T308L, I518N, L539P, K571E, D572V, N634F, K638I, K641V, A484C, T588C, and I542P, and / or a truncation after amino acid position 647 or amino acid position 695. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0113] In certain embodiments, the modified consensus sequence with HV loops modifications for HIV-1 CRF01_AE of SEQ ID NO: 105, or a variant thereof having at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105, includes one or more multiple founder variant modifications selected from H84N, N129S, N134K, G137N, N138T, S140E, G145S, E150K, K168P, N183S, K223S, N275G, N276S, A277V, G308S, E327D, N329I, T331N, K332Q, K332S, A341N, I354V, Q356T, P357L, M367I, K383R, N390S, G393K, S398G, G399N, G399E, G402R, V420D, R446A, G448S, G449E, G449N, N450S, N451G, N454K, N463F, T488S, R489S, R546K, E609R, N613G, N629D, N629S, D663E, R718S, G788E, L847I, and L852F, one or more repair modifications selected from S140T, E150D, K168Q, T331S, A341V, P357S, M367T, S398T, G449A, N451T, N463D, R489K, and E609D, one or more trimer stabilization modificationsAttorney Docket No.: HMJ-190-PCT selected from N298M, R300V, T314L, I524N, L545P, K577E, D578V, N640F, K644I, K647V, A490C, T594C, and I548P, and / or a truncation after amino acid position 653 or amino acid position 701. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0114] In some embodiments, the disclosure provides an immunogenic variant of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192 as disclosed herein, wherein the variant retains all of the modifications of the base sequence as summarized in Tables 4A-4C. For instance, an immunogenic variant of SEQ ID NO: 151 will retain all of the trimer stabilization modifications, the modification to the furin cleavage site, and the truncation after amino acid position 645 as provided in Table 4A. Similarly, an immunogenic variant of SEQ ID NO: 154 will retain all of the multiple founder variant modification, the repair modifications, the trimer stabilization modifications, the modification to the furin cleavage site, and the truncation after amino acid position 645 as provided in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 151, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 152, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 153, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, wherein the variant retains all of the modifications of the baseAttorney Docket No.: HMJ-190-PCT sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 155, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 156, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 157, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 158, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 159, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 160, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 161, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 162, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 163, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 164, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4A. In some embodiments, the variant has at least 90%Attorney Docket No.: HMJ-190-PCT sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 165, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 167, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 168, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 169, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 170, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 171, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 172, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 173, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 174, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQAttorney Docket No.: HMJ-190-PCT ID NO: 175, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 176, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 177, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 178, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4B. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 179, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 180, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 181, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 182, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 183, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 184, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 185, wherein the variant retains all of the modifications of the base sequence asAttorney Docket No.: HMJ-190-PCT summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 186, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 188, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 189, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 190, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 191, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 192, wherein the variant retains all of the modifications of the base sequence as summarized in Table 4C. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0115] In some embodiments, immunogenic fragments of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:Attorney Docket No.: HMJ-190-PCT 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192 are provided, including immunogenic fragments comprising one or more redesigned variable loop sequences disclosed in Table 1. In some embodiments, such immunogenic fragments include gp120, an outer domain, V1, V2, and / or V3. Typically, the immunogenic fragment retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0116] In some embodiments, variants of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192 are provided, including variants that retain immunogenic activity and variants comprising one or more redesigned variable loop sequences disclosed in Table 1. In some embodiments, such variants include gp120, an outer domain, V1, V2, and / or V3. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.

[0117] In some embodiments, the present disclosure provides a polynucleotide comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence of any one of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192, or an immunogenic fragment or variant thereof, optionally comprising gp120, an outer domain, V1, V2 and / or V3. Typically, the immunogenic fragment or variant thereof retains the glycan shield and optionally preserves the hypervariable loop anchor sites.Attorney Docket No.: HMJ-190-PCT Additional Modified HIV-1 Env Polypeptides

[0118] In certain embodiments, provided herein are modified HIV-1 Env polypeptides comprising one or more of: (1) multiple founder variant modifications, (2) repair modifications and trimer stabilization modifications, and / or (3) solubility / membrane-bound modifications as described herein elsewhere without redesigned HV loop(s). Such modified HIV-1 Env polypeptides can be obtained by introducing one or more of the aforementioned modifications into a naturally occurring HIV-1 Env polypeptide, such as one sampled from a patient infected by HIV- 1. In certain embodiments, the modified HIV-1 Env polypeptide further comprises one or more amino acid modifications in one or more of the hypervariable loops.

[0119] In some embodiments, such a naturally occurring HIV-1 Env polypeptide is from HIV- 1 subtype B and comprises the following amino acid sequence: MRVKGIKRNYQLWWRWGTMLLGMLMISSVAQQLWVTVYYGVPVWKEATTTLFCAS DAKAYDPEAHNVWATHACVPTDPSPLEIKLENVTEDYNMWKNNMVDQMHEDIISLWD QSLKPCVKLTPLCVTLDCTDYKGNTTKGNTKKGNTTGTPNNTGIGEMKNCTFNITSGIR GKMKKEYALFYTHDVIPIKEDSNSSTYRLISCNTSVITQACPKITFEPIPIHYCAPAGFAILK CNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEGIIIRSENISDNAKTIIVQLN KSVVINCTRPNNNTKKSIHFGPGRWNTVYATGAVVGDIRQAHCNISKKDWNDTLKQVV KKLREHFNKTIIFNQSSGGDLEVTMHTFNCGGEFFYCNTTQLFNSTWNSTQSLDNDTND TNHNITLPCRIRQIINMWQEIGKAMYAPPIRGQIRCSSNITGLLLTRDGGNNSSTNETFRPG GGNMKDNWRSELYKYKVVQIEPLGIAPTKARRRVVQREKRAVGIGALFLGFLGAAGST MGAAATTLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQARVLAIERYL RDQQLLGIWGCSGKLICPTNVPWNTSWSNRSGDDIWQNMTWMQWEREIDNYTGTIYR LLEESQTQQEKNEQELLALDKWANLWNWFNITNWLWYIKLFIMIVGGLIGLRIVFVVISI VNRVRQGYSPLSFQTHFPASRGPDRPGGIEEEGGERDRDRSRPLVDGFLALIWIDLRSLC LFSYHRLRDLLLIAARIVELLGRRGWESIKYLWNLLKYWGQELKNSAVSLYNAFAITVA EGTDRIIEVLLRIFRAICNIPTRIRQGAEKLLL (SEQ ID NO: 193).

[0120] In certain embodiments, the modified HIV-1 Env polypeptides without redesigned HV loop(s) of the disclosure are obtained by introducing into the naturally occurring HIV-1 Env polypeptide of SEQ ID NO: 193 one or more multiple founder variant modifications selected from R320K, I365L, I366T, G372N, T381S, R460W, L457N, R460T, R460G, R460S, G462H, G463D, G463V, G463A, N464I, N464E, S466I, S466T, S467N, S467A, N469S, N469G, G475K, N478D,Attorney Docket No.: HMJ-190-PCT N478S, G644K, G644E, N678G, N678Y, R748Y, and N813D, one or more repair modifications selected from L81Q, Y92F, D129N, T163S, S168T, G172D, H184L, V330I, I434V, G463A, G475A, N478D, A538S, I587V, G623L, N678S, and N678T, one or more trimer stabilization modifications selected from N309M, K311V, T327L, T539N, L560P, R592E, D593V, T655F, K659I, Q662V, A505C, P609C, and I563P, and / or a truncation after position 668 or position 716. Certain exemplified modified HIV-1 Env polypeptides without redesigned HV loop(s) derived from the naturally occurring HIV-1 Env polypeptide of SEQ ID NO: 193 are provided in Table 5 with the modifications made therein summarized in Table 6. Table 5. Exemplary Modified HIV-1 Env Polypeptides without Redesigned HV Loop(s) Derived from the Naturally Occurring HIV-1 Env Polypeptide of SEQ ID NO: 193 (“B036_env13.O2.Seed”). Name Sequence B036 env13.O2.Se MRVKGIKRNYQLWWRWGTMLLGMLMISSVAQQLWVTVYYGV T S T N P L I V T S T N P LAttorney Docket No.: HMJ-190-PCT YKYKVVQIEPLGIAPTKARRRVVQGGGGSGGGGSAVGIGALFLG FLGAAGSTMGAASNTLTVQARQLLSGIVQQQSNLPRAPEAQQH Q G V L F S T V E S F L V T S T P Q Q V TAttorney Docket No.: HMJ-190-PCT DKMKKEYALFYTLDVIPIKEDSNSSTYRLISCNTSVITQACPKITF EPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVS T P L S I V L F S T V T K F S R V T S T P LAttorney Docket No.: HMJ-190-PCT QLTVWGIKQLQARVLAVERYLEVQQLLGIWGCSGKLICCTNVP WNTSWSNRSLDDIWQNMTWMQWEREIDNYTETIYRLLEESQFQ V T S T P F Q S I V L F S T V N I Y S Q I I V T SAttorney Docket No.: HMJ-190-PCT TQLLLNGSLAEEGIIIRSENISDNAKTIIVQLNKSVVINCTRPNNMT VKSIHFGPGRWNTVYALGAIVGDIRQAHCNISKKDWNDTLKQV N P I V T S T N P G Q G V L F S T V N I G W E V P IAttorney Docket No.: HMJ-190-PCT KYLWNLLKYWGQELKNSAVSLYNAFAITVAEGTDRIIEVLLRIF RAICNIPTRIRQGAEKLLL (SEQ ID NO: 205) V T S T N P L L N EI V T S T N P L G Q GTable 6. Summary of Modifications Made to the Exemplary Modified HIV-1 Env Polypeptides without Redesigned HV Loop(s) Derived from the Naturally Occurring HIV-1 Env Polypeptide of SEQ ID NO: 193 Provided in Table 5 (Amino Acid Position Is Indexed by Referenced to SEQ ID NO: 193). Name SEQ MFV Repair Trimer Furin TruncationAttorney Docket No.: HMJ-190-PCT B036_e 194 L81Q, Y92F, N309M, 512-515: 668 nv13.O2 D129N, K311V, REKR-Attorney Docket No.: HMJ-190-PCT B036_e 200 R320K, I366T, L81Q, Y92F, N309M, 512-515: 668 nv13.O2 G372N, S467A, D129N, K311V, REKR-Attorney Docket No.: HMJ-190-PCT B036_e 206 G463A, S466T, L81Q, Y92F, N309M, 512-515: 668 nv13.O2 N469G D129N, K311V, REKR-

[0121] Accordingly, in certain embodiments, the disclosure provides an immunogenic variant of any one of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207 as disclosed herein, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 194, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 195, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 196, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 197, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In someAttorney Docket No.: HMJ-190-PCT embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 198, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 199, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 200, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 201, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 202, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 203, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 204, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 205, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 206, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. In some embodiments, the variant has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 207, wherein the variant retains all of the modifications of the base sequence as summarized in Table 6. Typically, the variant retains the glycan shield and optionally preserves the hypervariable loop anchor sites.Attorney Docket No.: HMJ-190-PCT

[0122] In some embodiments, immunogenic fragments of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207 are provided.

[0123] In some embodiments, the present disclosure provides a polynucleotide comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence of any one of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, or an immunogenic fragment or variant thereof. Nucleic Acids, Vectors, and Host Cells

[0124] In another aspect, the present disclosure is directed to an artificial nucleic acid molecule encoding any of the modified Env consensus sequences or immunogenic fragments or variants thereof disclosed herein, or a complementary sequence of the present isolated polynucleotides, or a vector comprising the same. The nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. The modified Env consensus sequences or immunogenic fragments or variants thereof disclosed herein can be synthesized as DNA sequences by standard methods known in the art and subsequently cloned and expressed in a recombinant host system using a suitable vector. The modified Env consensus sequences or immunogenic fragments or variants thereof disclosed herein can also be synthesized as RNA, such as messenger RNA (mRNA), sequences. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule (e.g., mRNA) with the specified sequence in which U, or a derivative thereof, such as pseudouridine, is substituted for T, unless context requires otherwise. Other nucleotide derivatives or modified nucleotides can be incorporated into the artificial nucleic acid molecules encoding the disclosed modified Env consensus sequences or immunogenic fragments or variants thereof. The synthesized DNA or mRNA sequences encoding the modified Env consensus sequences or immunogenic fragments or variants thereof can be codon-optimized so that expression of the encoded protein is improved and optimized for a particular expression system. Any codon optimization algorithms known in the art can be used to generate codon-optimized nucleic acid sequences.

[0125] A number of suitable vectors for expression of recombinant proteins in insect orAttorney Docket No.: HMJ-190-PCT mammalian cells are well-known and conventional in the art. Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., of mammalian origin or from a heterologous mammalian or non- mammalian species). In some embodiments, the vector is a plasmid or cosmid. In other embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral vector. In some embodiments, the vector can autonomously replicate in a host cell into which it is introduced. In some embodiments, the vector can be integrated into the genome of a host cell upon introduction into the host cell and thereby be replicated along with the host genome.

[0126] In some embodiments, particular vectors, referred to herein as “recombinant expression vectors” or “expression vectors”, can direct the expression of genes to which they are operatively linked. A polynucleotide sequence is “operatively linked” when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or regulatory DNA sequence is said to be “operatively linked” to a DNA sequence that codes for an RNA and / or a protein if the two sequences are operatively linked, or situated such that the promoter or regulatory DNA sequence affects the expression level of the coding or structural DNA sequence. Operatively linked DNA sequences are typically, but not necessarily, contiguous.

[0127] Accordingly, in some embodiments, provided herein are artificial nucleic acids encoding any of the modified Env consensus sequence or immunogenic fragment or variant thereof as disclosed herein. The artificial nucleic acids of the disclosure can be in form of a DNA or a RNA, such as a messenger RNA (mRNA). In some embodiments, the artificial nucleic acids of the disclosure are DNA molecules. In some embodiments, the artificial nucleic acids of the disclosure are RNA molecules. In certain embodiments, the artificial nucleic acids of the disclosure are mRNA molecules.

[0128] In some embodiments, the present disclosure is directed to a vector comprising the artificial nucleic acid molecule (e.g., mRNA) that encodes modified Env consensus sequence or immunogenic fragment or variant thereof as disclosed herein.

[0129] Generally, any system or vector suitable to maintain, propagate or express a polypeptide in a host may be used for expression of the modified Env consensus sequence orAttorney Docket No.: HMJ-190-PCT immunogenic fragment or variant thereof as disclosed herein. The appropriate DNA / polynucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001).

[0130] In another aspect, the present disclosure is directed to a host cell comprising any of the vectors disclosed herein including the expression vectors comprising the artificial nucleic acid molecules having the polynucleotide sequences encoding the modified Env consensus sequence or immunogenic fragment or variant thereof as disclosed herein. A wide variety of host cells are useful in expressing the present sequences. Non-limiting examples of suitable host cells include well-known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSCl, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells such as Expi293F cells and HOS cells.

[0131] Efficient expression of the present modified Env consensus sequences or immunogenic fragments or variants thereof as disclosed herein depends on a variety of factors such as optimal expression signals (both at the level of transcription and translation), correct protein folding, and cell growth characteristics. Regarding methods for constructing the vector and methods for transducing the constructed recombinant vector into the host cell, conventional methods known in the art can be utilized. While it is understood that not all vectors, expression control sequences, and hosts will function equally well to express the sequences of the present disclosure, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this disclosure. Immunogenic Compositions and Kits

[0132] Further disclosed herein are immunogenic compositions or vaccines comprising the modified Env consensus sequences, the modified HIV-1 Env polypeptides, or immunogenic fragments or variants thereof, of the present disclosure, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. The immunogenic compositions or vaccines can take any suitable form, including the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, and sustained-release formulations, injectants, andAttorney Docket No.: HMJ-190-PCT combinations thereof.

[0133] In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV-1 Env polypeptides derived from a single modified consensus sequence as disclosed herein, such as one of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, or SEQ ID NO: 118, or an immunogenic fragment or a variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV- 1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or a variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or a variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175,Attorney Docket No.: HMJ-190-PCT SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or a variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same.

[0134] In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV-1 Env polypeptides derived from a single naturally occurring HIV-1 Env polypeptide as disclosed herein, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise one or more modified HIV-1 Env polypeptides selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or a variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same. In certain embodiments, the immunogenic compositions or vaccines of the disclosure comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same.Attorney Docket No.: HMJ-190-PCT

[0135] The immunogenic compositions or vaccines disclosed herein comprising modified consensus sequences or immunogenic fragments or variants thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same, can be administered to a human patient, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The pharmaceutical compositions may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous, intramuscular, or subcutaneous administration of the modified consensus sequences or fragments or variants thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same, is preferred in certain embodiments. The immunogenic compositions or vaccines disclosed herein are administered to a patient or subject systemically, parenterally, or locally.

[0136] For parenteral administration, the modified consensus sequences or immunogenic fragments or variants thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same, can be formulated in a unit dosage injectable form (e.g., solution, suspension, or emulsion) in association with a pharmaceutically acceptable carrier.

[0137] Suitable pharmaceutically acceptable carriers include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). The compositions disclosed herein may be chosen from any suitable form, including injectable suspensions, solutions, sprays, lyophilized powders, syrups, and elixirs. Additional examples of carriers include water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin.Attorney Docket No.: HMJ-190-PCT Nonaqueous carriers such as fixed oils and ethyl oleate and liposomes may also be used. The carriers disclosed herein may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The modified consensus sequences or fragments or variants thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same, may be formulated in such carriers at concentrations of, for example, about 1 mg / ml to 10 mg / ml.

[0138] The vaccine or immunogenic composition disclosed herein may contain an adjuvant. As used herein, the term “adjuvant” refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. Adjuvants can include a suspension of minerals (alum, aluminum salts, including, for example, aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS) and / or potassium aluminum sulfate) on which antigen is adsorbed; or water -in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity.

[0139] Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as lipids and costimulatory molecules. Exemplary biological adjuvants include AS04 (Didierlaurent et al, J. Immunol., 2009, 183:6186-6197), IL-2, RANTES, GM-CSF, TNF- α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

[0140] The dose and dosage regimen may depend upon a variety of factors, such as the nature of the infection and the characteristics of the particular sequence or sequences to be administered, e.g., its therapeutic index, the patient, and the patient’s history. Generally, an amount of at least one modified consensus sequence or immunogenic fragment or variant thereof, or artificial nucleic acid molecules (e.g., mRNAs) encoding the same, is administered to a patient in an amount to induce an immune response in the patient. In particular embodiments, the amount of the modified consensus sequence or immunogenic fragment or variant thereof administered is in the range of about 0.1 mg / kg to about 20 mg / kg of patient body weight. Depending on the type and severity of the infection, about 0.1 mg / kg to about 20 mg / kg body weight (e.g., about 0.1-15 mg / kg / dose) of antigen is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of the therapy mayAttorney Docket No.: HMJ-190-PCT be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[0141] In some embodiments, the vaccines of the disclosure are mRNA vaccines comprising one or more mRNAs encoding the modified consensus sequence or immunogenic fragment or variant thereof disclosed herein. In some embodiments, the one or more mRNAs are encapsulated in a lipid nanoparticle. In some embodiments, the mRNA vaccine of the present disclosure contains at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg, including all values and subranges therebetween, of encapsulated mRNA. In some embodiments, the mRNA vaccine of the present disclosure contains about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, or at least about 1000 μg, including all values and subranges therebetween, of encapsulated mRNA.

[0142] In yet another embodiment, there is provided a kit for performing diagnostic and prognostic assays using the modified consensus sequences or immunogenic fragments or variants disclosed herein. Kits may include a suitable container comprising at least one modified consensus sequence or immunogenic fragment or variant thereof in either labeled or unlabeled form. In addition, when the modified consensus sequences or immunogenic fragments or variants are supplied in a labeled form suitable for an indirect binding assay, the kit further includes reagents for performing the appropriate indirect assay. For example, the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and / or instructions may also be included. Methods

[0143] In certain embodiments of all aspects of the disclosure, the modified consensus sequences or immunogenic fragments or variants thereof disclosed herein are used in a method of detecting binding of HIV-1 antibodies to antigens, such as Env. Such methods may include, but are not limited to, antigen-binding assays that are well-known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry. In certain embodiments disclosed herein, the binding capacity of the antibodies to the modified consensus Env may be evaluated by cell-based ELISA.

[0144] In certain embodiments, disclosed herein is a method of inducing an immune responseAttorney Docket No.: HMJ-190-PCT comprising administering to a subject a vaccine or immunogenic composition comprising at least one modified consensus sequence or immunogenic fragment or variant thereof as disclosed herein. Also disclosed is a vaccine or immunogenic composition as disclosed herein for use in a method of inducing an immune response in a subject. Also disclosed is an immunogenic composition as disclosed herein for the manufacture of a vaccine for use in a method of inducing an immune response in a subject. The immune response can be induced in a naïve subject who has not previously been exposed to HIV or in a subject who is at risk for HIV-related disorders. Alternatively, the immune response can be induced in a subject who has been previously exposed to HIV and used to enhance an existing immune response.

[0145] In some embodiments, the method of inducing an immune response (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or immunogenic fragments or variants thereof. In some embodiments, the method of inducing an immune response (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or immunogenic fragments or variants thereof. In some embodiments, the method of inducing an immune response (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV- 1 Env polypeptides of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or immunogenic fragments or variants thereof. In some embodiments, the method of inducing anAttorney Docket No.: HMJ-190-PCT immune response (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or immunogenic fragments or variants thereof,

[0146] In the methods or uses disclosed herein, the immune response can be measured using routing methods in the art, such as those disclosed in this application. These routine methods include, but are not limited to, measuring antibody response, such as an antibody response directed against an HIV-1 Env protein.

[0147] In certain embodiments, there are further disclosed herein methods of treating or preventing HIV infection comprising administering to a subject a vaccine or immunogenic composition comprising at least one modified consensus sequence or immunogenic fragment or variant thereof as disclosed herein. Also disclosed is a vaccine or immunogenic composition as disclosed herein for use in a method of treating or preventing HIV infection in a subject. Also disclosed is an immunogenic composition as disclosed herein for the manufacture of a vaccine for use in a method of treating or preventing HIV infection in a subject.

[0148] In some embodiments, the method of treating or preventing HIV infection (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or immunogenic fragments or variants thereof. In some embodiments, the method of treating or preventing HIV infection (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ IDAttorney Docket No.: HMJ-190-PCT NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or immunogenic fragments or variants thereof. In some embodiments, the method of treating or preventing HIV infection (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or immunogenic fragments or variants thereof. In some embodiments, the method of treating or preventing HIV infection (or related uses thereof) comprises administering to a subject a pharmaceutical composition comprising at least one, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 of the modified HIV-1 Env polypeptides of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or immunogenic fragments or variants thereof.

[0149] Subjects at risk for HIV-related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to HIV in some way. Administration of an immunogenic composition or vaccine comprising at least one modified consensus sequence or immunogenic fragment or variant thereof as disclosed herein can occur prior to the manifestation of symptoms characteristic of HIV-related disease or disorder, such that a disease or disorder is prevented or delayed in its progression.

[0150] In certain aspects, the methods of treating or preventing HIV infection (or related uses thereof) may further comprise co-administering other agents suitable for the treatment of HIV infection, such as anti-retroviral therapies. Anti-retroviral therapies that may be co-administered with the modified consensus sequences or fragments or variants thereof disclosed herein may include, for example, nucleoside analog reverse-transcriptase inhibitors (such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and apricitabine), nucleotide reverse transcriptase inhibitors (such as tenofovir and adefovir), non- nucleoside reverse transcriptase inhibitors (such as efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine), protease inhibitors (such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir,Attorney Docket No.: HMJ-190-PCT lopinavir, fosamprenavir, atazanavir, tipranavir, and darunavir), entry or fusion inhibitors (such as maraviroc and enfuvirtide), maturation inhibitors (such as bevirimat and vivecon), or broad spectrum inhibitors, such as natural antivirals.

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES Methods for Hypervariable Loop Analysis

[0152] The following methods were used in conducting the studies described in the examples that follow.

[0153] Dataset: Env amino acid sequences of subtypes B, C, and CRF01_AE were retrieved from the Los Alamos National Laboratory (LANL) HIV-1 sequence database on 7 November 2022. Sequences were retained if they had a complete open reading frame, information on the year and location of collection, and were not hypermutated or problematic (as defined by LANL). Non-independent sequences were removed so that the final dataset included one sequence per person. The IC50of PGT145, VRC26.25, 10-1074, PGT135, VRC01, and 3BNC117 against viruses that had the corresponding Env sequences available were downloaded from the CATNAP database (Yoon et al., Nucleic Acids Res., 2015, 43:W213-W219) as of 4 October 2022. In total, there were 549, 423, 782, 310, 1032, and 818 IC50s and associated Env sequences for PGT145, VRC26.25, 10-1074, PGT135, VRC01 and 3BNC117, respectively.

[0154] HV loop analysis: The HXB2 genome annotation from the LANL HIV Database, available online on the world wide web at hiv.lanl.gov, was followed to define the start and end of HV loops, which were located at 132-152, 185-190, 396-410 and 460-467, for V1HV, V2HV, V4HV and V5HV, respectively. As an exception, V4HV was extended to include 395 and 411- 412 anchor sites in the HV loop redesign. Structurally, the distance between the start and end of spacers was calculated as the distance between CA atoms before the N-terminal and after the C-Attorney Docket No.: HMJ-190-PCT terminal of the spacer loop. The HXB2 sequence was added and aligned to define the positions. Multiple Alignment using Fast Fourier Transform (MAFFT) was used to align sequences. For the logo plot of HVs, HV loops were split from the middle. The first half was left-aligned to the start position while the second half was right-aligned to the end position.

[0155] Structure Prediction and Analysis: ColabFold, which integrated AlphaFold-Multimer with other tools and databases, was used to model unmodified and redesigned Env structures. During the prediction, one of the three subunits in the prefusion Env trimer was predicted by feeding one gp120 (31-511) and one gp41 (512-665) as input. The top model from five predictions was adopted for later analysis. Loop modeling was done with the KIC loop modeling protocol (Stein et al., PloS One, 2013, 8:e63090) via PyRosetta. The four HV loops were modeled sequentially in random orders in individual runs.

[0156] The non-HV surrounding surface sites of HV loops were defined as Env sites that were not part of the HV loop which had a relative accessible surface area (RSA) greater or equal to 0.30 and had any atoms within 10 Å of any HV loop atoms in the predicted unmodified consensus B Env structure. The accessible surface area (ASA) was approximated by the Shrake-Rupley method. In brief, evenly distributed points (n=256) were placed on spheres that were centered at atoms in the protein. The radius of the spheres was the sum of the atoms’ and the solvent probe’s radius (1.5 Å). The accessible surface was defined after filtering out points on a sphere that were within neighboring spheres. Then, the RSA of a site was calculated as the ratio of ASA over the estimated maximum ASA. The same approach was also used to estimate depth, only the probe radius was set to 30 Å, and the depth was equal to the minimal distance between a protein atom and the accessible surface minus the probe radius. Env structure 5FYJ (Stewart-Jones et al., Cell, 2016, 165:813-826) was used for the ASA estimation. It was also used for the illustration of HV loop and nearby bnAb epitopes in FIG. 1A.

[0157] The clashes between antibody and Env was determined as the number of antibody atoms within 2.5 Å of Env after superimposing the modeled Env on the Env in the solved Env- antibody complex. Bio.PDB (Hamelryck et al., Bioinforma. Oxf. Engl., 2003, 19:2308-2310) in Biopython (Cock et al., Bioinforma. Oxf. Engl., 2009, 25:1422-1423) was used to superimpose structures. The Env-antibody complexes used, listed as PDB code, were 5V8L (Lee et al., Immunity, 2017, 46:690-702), 6VTT (Gorman et al., Cell Rep., 2020, 31:107488), 6UDJ (Schommers et al., Cell, 2020, 180:471-489.e22), 4JM2 (Kong et al., Nat. Struct. Mol. Biol., 2013,Attorney Docket No.: HMJ-190-PCT 20:796-803), 6V8X (Henderson et al., Nat. Commun., 2020, 11:520), and 7PC2 (Lorin et al., J. Exp. Med., 2022, 219:e20212045) for PGT145, VRC26.25, 10-1074, PGT135, VRC01 and 3BNC117, respectively. PyMol (Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 1.8. (2015)) was used for the structure visualization and figure generation. Hydrogen atoms were removed from the structure, if there was any, in all analysis.

[0158] Protein Production and Antibody Binding: Uncleaved gp140 proteins were designed with R to S mutations in the gp120 / gp41 furin cleavage site and incorporated the native leader peptide and full MPER sequence, followed by a short GGGS (SEQ ID NO: 215) linker sequence and a C-terminal AviTag. Sequences, codon-optimized for expression in human cells, were synthesized (Genscript) and cloned into a custom pcDNA3.4 (ThermoFisher) expression vector. Proteins were expressed by transient transfections in Expi293F cells (ThermoFisher) according to the manufacturer’s instructions. Gp140 proteins were purified from clarified cell culture supernatants 4 days post-transfection using Galanthus nivalis lectin (GNL) affinity chromatography followed by a Q-sepharose (GE Healthcare) polishing step to remove host cell protein contaminants. All gp140s were purified to 90% purity or higher, as assessed by SDS- PAGE and Coomassie staining in reducing and non-reducing conditions. Antibodies VRC01, 3BNC117, CD4-Ig, PG9, PGDM1400, PGT128, 2G12, 10E8, and 4E10 were produced in-house from public sequences as IgG1 by transfection in Expi293F cells and purified by protein A affinity chromatography; antibodies 447-52D and VRC42.01 were obtained from Polymun (Cat. No. AB014) and from Nicole Doria-Rose (VRC, NIH), respectively. For antibody binding and characterization, gp140 proteins were coupled to uniquely coded carboxylated magnetic microspheres (Luminex Corp., Austin TX) per manufacturer’s protocol and as previously described and pooled into an antigen cocktail. Antibody binding and characterization were performed as previously described (Mdluli et al., PLoS Pathog., 2020, 16:e1009101; Tomaras et al., J. Virol., 2008, 82:12449-12463; Brown et al., J. Immunol. Methods, 2012, 386:117-123) with minor modifications. The antigen cocktail was incubated with an array of heat-inactivated human plasma pool samples from various clade-specific cohorts of people living with HIV-1, or with monoclonal antibodies targeting various regions of HIV-1 Env. Samples were incubated for 2 hours at room temperature, washed then incubated for 1 hour with phycoerythrin labeled anti- human IgG Fc (Southern Biotech, Birmingham AL) detection antibody. After a final wash to remove unbound detection reagent, microspheres were resuspended in 40 μL sheath fluidAttorney Docket No.: HMJ-190-PCT (Luminex Corp) and data was collected on a BIO-PLEX®3D Suspension Array system (Bio-Rad, Hercules CA) running xPONENT® v.4.2 (Luminex Corp). A multi-clade control plasma pool comprised of samples from HIV-1 Group O, subtypes B, C, and CRF01_AE, CRF02_AG, and CRF03_AB (SeraCare, Milford MA) was used as a positive control (“Positive Pool”) and a pool of plasmas from people without HIV-1 served as a negative control. The figures correspond to monoclonal antibodies at a concentration of 10 µg / mL and pooled plasma at 1:100 dilution. Responses are reported as fold-over people without HIV-1 for plasma samples and fold-over the ZIKA virus-specific monoclonal antibody MZ4.

[0159] HIV-1 Env Expression and Antibody Binding: 293T cells were transfected with 2 µg of naked mRNA encoding an HIV-1 env gene (jetMESSENGER, mRNA transfection reagent, (VWR, Cat. No. 75806-224) in 6-well plates and harvested at 48  hours. Cells were split into separate staining reactions for monoclonal antibodies 3BNC117 (BEI Resources, Cat. No. ARP- 12474), 10-1074 (BEI Resources, Cat. No. ARP-12477), 10E8 (BEI Resources, Cat. No. ARP- 12294), 447-52D (BEI Resources, Cat. No. ARP-4030), b12 (Polymun, Cat. No. AB011), PGDM1400 and PGT151 (both kindly provided by Dr. Devon Sok and International AIDS Vaccine Initiative). 2G12 was produced in Expi293F cells from publicly available heavy and light chain sequences (Calarese et al., Science, 2003, 300:2065-2071) and purified using Protein A affinity chromatography. Cells were washed and stained with anti-human IgG-PE at 1:250 dilution (SouthernBiotech, Cat. No. 2040-09) for 15 minutes and fixed in 0.5% paraformaldehyde. Fluorescence was measured on a FACSymphony A5 SE Cell Analyzer and analyzed in FlowJo v.9.9.6.

[0160] Recombinant HIV-1 Env Stabilized Trimer Production and Purification: Stabilized, soluble trimers in a prefusion state were engineered by introducing previously reported (Rutten et al., Cell Rep., 2018, 23:584-595; Rawi et al., Cell Rep., 2020, 33:108432) and additional modifications with ten stabilization mutations (M302, V304, L320, N535, P556, E588, V589, F651, I655, and V658) and three SOSIP mutations (C501, C605 and P559). Sites 508-511 were replaced with six arginines for more efficient furin cleavage. All Envs were terminated at site 664. Trimers were expressed in Expi 293 cells (Thermo Fisher Scientific) by transient transfection with plasmid DNA complexed with PEI. 800 μg of the HIV-1 Env expressing plasmid and 200 μg of furin expressing plasmid were used for each 1 L of cells. Cell-free culture supernatant was 0.2 μm filtered and purified by GNA Lectin (VectorLabs) before size exclusion chromatography on aAttorney Docket No.: HMJ-190-PCT Superdex 20016 / 600 column (GE Healthcare Cat. No. 29323952) in PBS. Any shoulders to the roughly monodisperse Env trimer peak were removed, and protein was pooled and concentrated before adding glycerol to a final concentration of 5%. Trimeric HIV-1 Env protein was sterile- filtered, snap-frozen, and stored at -80 °C before further use.

[0161] Negative-stain Electron Microscopy: Purified protein was thawed and diluted to 10 µg / ml with 20 mM HEPES, pH 7.5; 150 mM NaCl immediately prior to application on glow- discharged carbon-coated grids (Electron Microscopy Sciences #CF300H-Cu-50) and stained with 2% phosphotungstic acid, pH 7.4. Samples were imaged on a JEOL CryoARM 200 using SerialEM 4.1.0beta with a nominal magnification of 60kX and a pixel size of 0.873 Å / pixel on a Ametek Gatan K3 in counting mode. A 100-frame movie with a total dose of 225 electrons / Å2was motion corrected and CTF was estimated using patch CTF in CryoSPARC v4. All further processing was done in Cryosparc v4. Particle picking was conducted with a circular Gaussian blob of 100-150 Å for all samples. Reference-free 2D classification was used to separate false picks from putative particles. A second round of 2D with a maximum alignment resolution of 15 Å and a maximum resolution of 10 Å on putative particles was used to separate monomer and trimer classes and estimate the relative percentages of each species. For the gp140 samples, no averageable trimer was observed.

[0162] For stabilized trimer samples, A C1 ab initio model was created for particles that classified as trimers in 2D for each of the protein samples imaged. This was used as an initial model for a C13D homogenous refinement with an initial low pass filter of 60 Å, GS-FSC split of 40 Å, and a maximum alignment resolution of 20 Å. The resulting consensus structure was 3D classified without alignment with 10 classes and a target resolution of 15 Å. The particles from the most open trimer class and most closed trimer class were selected and refined with 3D homogenous refinement using the volume from their classification with the same parameters as noted above in both C1 and C3. C1 and C3 closed and open models were then used as initial models for a two-class heterogenous refinement with an initial resolution of 30 Å and a maximum resolution of 15 Å. The relative percentages and maps shown were estimated using this C1 heterogeneous refinement, but C3 refinements converged on the same approximate proportions.

[0163] This procedure was effective for all samples except for the subtype C consensus stabilized trimer without HV loop redesign. In this case, the classification without alignment did not produce a particle subset that was closed enough to separate closed from open trimer usingAttorney Docket No.: HMJ-190-PCT heterogenous refinement. In this case, an initial model of closed trimer from the subtype C stabilized trimer with HV loop redesign sample and an open trimer without HV loop redesign was used for the final C1 heterogenous refinement. Data was visualized and figures were generated using UCSF ChimeraX and EMAN2.

[0164] HIV-1 Pseudovirus (PSV) Production, Titration and Neutralization: HIV-1 PSVs were produced by co-transfection of HEK293T / 17 cells (ATCC, Manassas, VA, USA) with a pSG3ΔEnv DNA plasmid encoding the HIV-1 backbone (24 μg DNA) and a plasmid encoding the HIV-1 env gene (8 μg DNA). Plasmids were combined with X-tremeGENE 9 DNA transfection reagent (Sigma-Aldrich, St. Louis, MO, USA) at a 1:3 ratio in serum-free DMEM, per manufacturer recommendations. The transfection mix was added to a T75 flask containing plated HEK293T / 17 cells in a culture medium. The flask was incubated for 6 hours at 37°C. Then the media was replaced. PSVs were harvested 72 hours after transfection, filtered through a 0.45 μm PVDF membrane (MilliporeSigma, Burlington, MA, USA), and stored in 3 ml aliquots at -80°C.

[0165] PSV stocks were titrated on TZM-bl cells (NIH AIDS Research and Reference Reagent Program, Cat. No. 8129) to determine an acceptable dilution for neutralization assays. Virus stocks were serially diluted then added to 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) in a final 25 μL / well volume. An additional 25 μL of supplemented DMEM was added to each well. TZM-bl cells were added to plates in 50 μL of supplemented DMEM with 40 μg / mL of DEAE-Dextran (Sigma, St. Louis, MO, USA). Virus and cells were incubated together for 48 hours at 37°C. Infectivity was measured by luminescence after adding 100 μL of the Bright- Glo Luciferase Assay System (Promega, Madison, WI, USA) to each well; luminescence was quantified using the EnVision luminometer (Perkin Elmer, Waltham, MA, USA).

[0166] Neutralizing antibody titers were determined using TZM-bl cells in a manual 96-well assay. BnAbs were obtained from the HIV reagent Program (VRC01 (Cat. No. ARP-12033, Lot 180231); 3BNC117 (Cat. No. ARP-12474, Lot 140211); sCD4 (Cat. No. ARP-4615, Lot 180011); PGT145 (Cat. No. ARP-12703, Lot 190527); 10-1074 (Cat. No. ARP-12477, Lot 190442); PGT128 (Cat. No. ARP-13352, Lot 190522); 2G12 (Cat. No. ARP-1476, Lot 180138); 10e8 (Cat. No. ARP-12294, Lot 170395) or from PolyMun (PG9 (Cat. No. AB015, Lot PG9_021014A); 4e10 (Cat. No. AB004, Lot T531002-A)). BnAbs were prepared at a starting concentration of 25 μg / mL in supplemented DMEM. PSVs were diluted to a concentration to yield 100,000 relative light units (RLUs) and at least 3 times above the cell-only controls, as determined by the PSV titration assay.Attorney Docket No.: HMJ-190-PCT Neutralization reagents were serially diluted. Titered bNAbs were transferred to 96-well culture plates and incubated with an equal volume of PSV for 1 hour at 37°C. A non-specific virus control (MuLV) was used to ensure neutralization by the bNAbs was HIV-specific. TZMbl cells (1 × 104cell / well) mixed with 40 μg / mL DEAE-dextran (Sigma, St. Louis, MO, USA) were added to each well and incubated for an additional 48 hours. RLUs were detected with the EnVision luminometer (Perkin Elmer, Waltham, MA, USA) using the Bright-Glo Luciferase Assay System (Promega Corporation, Madison, WI, USA). Percent neutralization (percentage reduction of relative light units in the presence of bNAb) was calculated for each bNAb concentration. Neutralization dose-response curves were fitted by nonlinear regression using the LabKey Server, and the final titer is reported as the concentration of bNAb (IC50 / IC80) necessary to achieve 50% or 80% neutralization.

[0167] Quantification and Statistical Analysis: Statistical analyses were performed using SciPy (Virtanen et al., Nat. Methods, 2020, 17:261-272) and coin (Hothorn et al., Am. Stat., 2006, 60:257-263). The non-paired, non-parametric Mann-Whitney U tests were used to compare the IC50 of short / long nearby or distant HV loops. The paired, non-parametric Wilcoxon signed-rank tests were used to compare the depth difference before and after HV loop redesign and the binding responses before and after the HV loop redesign with pooled plasma samples or bnAbs. The one- tailed exact Wilcoxon-Pratt signed-rank test was carried out using the coin package (Hothorn et al., Am. Stat., 2006, 60:257-263). Differences corresponding to p-values of less than 0.05 were considered as significant. Example 1. Analysis of Short HV Loops for HIV-1 Subtypes B, C, and CRF01_AE

[0168] Short HV Loops Were Rare for Subtypes B, C, and CRF01_AE: publicly available circulating HIV-1 sequences were curated to obtain a dataset of 4,847 independent Env sequences sampled from people living with HIV between 1979 and 2020. These sequences corresponded to HIV-1 subtype B (n=2495), subtype C (n=1503) and CRF01_AE (n=849). The distribution of HV loop lengths was analyzed as well as the number of potential N-linked glycosylation sites (PNGS) in four HV loops. The HV loops corresponded to a portion of the variable loops: V1HV, sites 132- 152 within sites 131-157 (V1); V2HV, sites 185-190 within sites 157-196 (V2); V4HV, sites 395- 412 within sites 385-418 (V4); and V5HV, sites 460-467 within sites 460-470 (V5). V3 was omitted because it does not contain an HV segment. The location of the HV loops, as well asAttorney Docket No.: HMJ-190-PCT adjacent bnAb epitopes, are shown on the structure in FIG. 1A.

[0169] V1HV loops showed a median length of 20 (subtype C) to 23 (CRF01_AE) amino acids. V1HV loops were about twice as long as other HV loops, which had a median length ranging between 7-9 amino acids (V2HV), 9-12 amino acids (V4HV), and 8-9 amino acids (V5HV). The length distributions across subtypes / CRF were similar for V2HV and V5HV, while there were differences for V1HV and V4HV. CRF01_AE showed fewer short V1HV loops (25th percentile=22 amino acids), than subtype B (19 amino acids) or subtype C (16 amino acids). Subtype B had fewer short V4HV loops (25th percentile=11 amino acids) than subtype C (6 amino acids) and CRF01_AE (7 amino acids). Similar patterns were observed for the number of PNGS on HV loops across subtypes / CRF. Sequences with V1, V4, and V5 HV loops that were shorter than or equal to the 10th percentile were very rare representing 0.73%, 0.53%, and 0.32% of the dataset for subtypes B, C, and CRF01_AE, respectively. The loop lengths of the sequences that have been used as inserts in previous vaccine efficacy or are considered for future trials were also determined. It was found that these insert sequences usually had one or more long HV-loops. For example, the subtype C TV1 sequence used in the vaccine in HVTN100 and HVTN702 efficacy trial had a 33-amino acid long V1HV (the median V1HV for subtype C is 20 AA), which is about three times longer than the length of the redesigned V1HVs (8 to 13 amino acids). Bekker et al., Lancet HIV, 2018, 5:e366-e378; and Gray et al., N. Engl. J. Med., 2021, 384:1089-1100. Mosaic2.Env in the Mosaico study (Barouch et al., Nat. Med., 2010, 16:319-323) had a 16-amino acid long V2HV that is twice as long (16 amino acids) as the median for subtypes B and C (7 and 9 amino acids, respectively) and three times longer than the designed V2HVs (4 to 5 amino acids). Some exceptions with short HV loops were also identified, such as the subtype A192RW02041 insert that was part of the trivalent vaccine tested in the HVTN505 trial and the subtype C CH50544 insert that has been undergoing early clinical testing. Example 2. Analysis of Long HV Loops and Sensitivity to bnAbs

[0170] The neutralization sensitivity to six bnAbs was compared for HIV-1 Env sequences with the shortest (≤ 5 percentile) and longest (≥ 95 percentile) HV loops. All neutralization and corresponding sequence data were obtained from CATNAP (Yoon et al., Nucleic Acids Res., 2015, 43:W213–W219). The bnAbs belonged to three representative epitope groups: (1) V2-apex antibodies PGT145 and VRC26.25; (2) glycan-supersite antibodies 10-1074 and PGT135; and (3)Attorney Docket No.: HMJ-190-PCT CD4bs antibodies VRC01 and 3BNC117. To evaluate the effect of loop length on bnAb sensitivity, short and long HV loops for the HV loop proximal to each bnAb (V2HV to V2-apex antibodies, V1HV to glycan supersite antibodies, and V5HV to CD4bs antibodies) and for the three other HV loops were compared. All neutralization and corresponding sequence data were obtained from CATNAP (Yoon et al., Nucleic Acids Res., 2015, 43:W213-219). As shown in FIG.3A-3F, the virus sequences with the longest adjacent HV loops had significantly higher half maximal inhibitory concentration (IC50) than the sequences with the shortest HV loops (p-value ≤ 0.015, one-tailed Mann-Whitney U test) for the six bnAbs, suggesting that longer HV loops obstruct bnAb epitope availability. When HV loops distant from the bnAb epitope (V5HV to V2- apex and glycan supersite antibodies and V2HV to CD4bs antibodies) were compared, there was no significant difference in sensitivity for short or long HV loops for five of the six bnAbs tested (p-value ≥ 0.207). The results for PGT145 are shown in FIG. 3A, VRC26.25 are shown in FIG. 3B, 10-1074 are shown in FIG. 3C, PGT135 are shown in FIG. 3D, VRC01 are shown in FIG. 3E, and 3BNC117 are shown in FIG. 3F.

[0171] Interestingly, there might be some longer-range interactions between HV loops. Sequences with long / short V1HV had a similar impact on IC50 values for V2-apex antibodies as V2HV, but there is no association between the length of V1HV and V2HV (data not shown), suggesting that interactions between V1HV and V2HV may also modulate the accessibility of V2- apex epitopes. For VRC01, sequences with shorter HV loops for V1, V2, and V5 were significantly associated with increased sensitivity when compared to sequences with longer HV loops, as shown in FIG. 3E. An exemplary graphical illustration of interference from long HV loops on the binding potential of bnAbs that target adjacent sites, as well as the improvement in accessibility afforded by short HV loops, is shown, for example, in FIG. 1B.

[0172] The strongest impact was seen for V1HV loops and the neutralization sensitivity to 10- 1074. HIV-1 Envs with the shortest V1HV loops had a median IC50 greater than or equal to 5,000 times lower than the strains with the longest V1 HV loops (0.02 μg / ml vs. ≥ 100 μg / ml, one-tailed Mann-Whitney U test p-value 0.00003). For V5HV loops, which are distant from the 10-1074 epitope, there was no significant difference in sensitivity when short and long loops were compared (one-tailed Mann-Whitney U test p-value 0.838). These data masked some subtype-specific differences, as differences between short and long HV loops were not manifest for subtype B (with p-values ≥ 0.139) except for 10-1074 (p = 3.6e−07). Differences for subtype C were generallyAttorney Docket No.: HMJ-190-PCT statistically significant, except for 10-1074 (p=0.129) and PGT135 (p=0.128). The analysis was repeated using 10–25% as the threshold to compare the extremes of the distributions. Differences between short / long adjacent HV loops remained statistically significant for most bnAbs (p < 0.045) or borderline significant for VRC26.25 when the cutoff was set as 25% (p = 0.079), with PGT145 as an exception (p ≥ 0.101 with 10-25% as the threshold). Overall, these results indicated that a long HV loop can diminish the virus’s sensitivity to a bnAb. Example 3. Redesign of HV Loops

[0173] The HV-loops on Env were redesigned to improve accessibility to typical bnAb epitopes. Env consensus sequences which were derived only from sequences that were sampled after 2010 were used, thus reflecting currently circulating viruses. The redesign was done for subtypes B, C, and CRF01_AE. These subtypes were selected in part because large datasets of sequences were available. In addition, HIV-1 subtype B is the most thoroughly characterized subtype, subtype C is responsible for the majority of cases among people living with HIV, and CRF01_AE is the virus that was circulating in a HIV-1 vaccine trial where some efficacy was recorded. The goals of this study were to: (1) make the loop as short as possible while maintaining structural integrity; (2) preserve the loop anchor sites, which are the relatively conserved start and end of the HV loops that form favorable interactions with the non-HV part of Env; and (3) maintain the glycan shield intact by ensuring there was at least one PNGS on each redesigned loop. To apply this strategy to the V1HV loop of subtype B, the sequence alignment and the AlphaFold2 predicted structure of the corresponding consensus sequence were analyzed. The relatively conserved TDL motif (sites 132-134) as N-anchor sites was identified, as well as the relatively conserved MEKG (SEQ ID NO: 121) motif (sites 149-152) as C-anchor sites, with all sites in between considered spacer sites. In the TDL motif, D133 may form favorable charge-charge interactions with K151 in the C-anchor sites or with K155; L134 may form hydrophobic interactions with I154, L175, I323, and I326. In the MEKG (SEQ ID NO: 121) motif, M149 may form hydrophobic interactions with I326 and I154; E150 may form a favorable charge-charge interaction with R419. These anchor sites were retained since they did not have direct contact with glycan supersite antibodies. Different spacer lengths were tested iteratively using AlphaFold2 predicted structures by comparing the predicted local Difference Distance Test (lDDT) scores, which measure the confidence given by AlphaFold2 for each output structure. A specific spacerAttorney Docket No.: HMJ-190-PCT was selected when it maintained or increased the predicted lDDT for the anchor and surrounding sites of the HV loop, suggesting that the anchor stability will be maintained. For V1HV, the spacer was shortened from 15 amino acids to 5 amino acids. This resulted in the V1HV loop length changing from 22 amino acids (50thpercentile in subtype B sequences) to 12 amino acids (4thpercentile in subtype B sequences). In addition, for subtype B, V2HV was modified from 7 to 4 amino acids, and V5HV was modified from 7 to 5 amino acids. The V1HV, V2HV, and V5HV loops of subtypes B, C, and CRF01_AE were redesigned with the same strategy, and the corresponding redesigned hypervariable loop sequences are shown in Table 1.

[0174] Contrary to the strategy for V1HV, V2HV, and V5HV loops, for V4HV loops, the shortest V4HV loops to accommodate its specific structural context was not sought. Sites adjacent to V4HV (non-HV sites within 10 Å of the HV loops, relative surface area > 0.30) were diverse, and no major bnAbs are known to target this segment of Env. For subtype C, the median Shannon entropy of V4HV adjacent sites was 2.4 bits compared to 0.8, 0.7, and 1.1 bits, respectively, for the sites adjacent to V1HV, V2HV and V5HV. This high Shannon entropy for V4HV loops indicated that there were 4.5 (22.4 / 1.1) to 10.8 (22.4 / 0.7) times more amino acid possibilities on average for surface sites adjacent to V4HV compared to the other HV loops. In addition, the distance encompassing spacer sites was larger for V4HV than for V1HV, V2HV, and V5HVs (20.8 Å for V4HV, versus 10.8, 7.9, and 9.4 Å for V1HV, V2HV and V5HV, respectively, based on the AlphaFold2 model of the subtype B consensus). This suggests that a short V4HV spacer may not be compatible with the structure. Hence, a medium length was chosen for the V4HV loop; this loop length could also partially shield the highly diverse segment adjacent to the V4HV. For the subtype B consensus, W395 was identified as the N-terminal anchor site, as it is well conserved and forms hydrophobic interaction with nearby non-HV sites, and ND (sites 411-412) was identified as the C-terminal anchor sites, as N is the most frequent option and D / E412 might form a charge-charge interaction with R335. The spacer was replaced with an 11-amino acid linker. The redesigned linker contained two PNGS, which was the median number for V4HV subtype B, C, and CRF01 sequences. The V4HV loops of subtype C and CRF01_AE were redesigned with the same strategy. One notable feature of the V4HV redesign for CRF01_AE is that C395 (which was predicted to form a disulfide bond in the redesigned sequence) was replaced with the second most frequent residue at that site, W, and the spacer was extended to better shield the surrounding sites of V4HV. Across all redesigned HV loops, the spacer consisted mainly of glycine and serineAttorney Docket No.: HMJ-190-PCT which lack the side chains that are preferentially targeted by antibodies and may also increase the flexibility of the HV loops. The redesigned HV loops tended to have higher predicted IDDT scores from AlphaFold2 than the unmodified ones, except for the V4HV of the subtype C consensus. All redesigned HV loops are listed in Table 1.

[0175] To measure if the redesign improved accessibility of bnAb epitopes, a depth index was calculated to correspond to the closest distance between an epitope site and the antibody probe. The antibody probe is a sphere with a radius corresponding to an antibody's Fv (=30 Å) that rolls around the Env structure, as illustrated in FIG. 2A. A short depth indicates that the site is more easily accessible to the antibody. The depth corresponding to the epitopes of two glycan supersite bnAbs, 10-1074 and PGT135, and two CD4bs bnAbs, VRC01 and 3BNC117, before and after the HV loop redesign was calculated. Each loop was remodeled 500 times via the kinematic closure (KIC) loop modeling protocol from PyRosetta. Among the 500 models, the 100 models with the lowest Rosetta energy were selected for the epitope depth calculation. The epitope depth decreased for the four bnAb epitopes based on the structural model of the Env consensus of subtypes B, C, and CRF01 AE, as shown in FIG.2B-2D. The median depth decreased by 0.8 to 1.6 Å for glycan supersite antibody epitope sites, while the median depth decreased by 0.2 to 0.8 Å for CD4bs antibody epitope sites. Besides the depth, the clashes between HV loops and corresponding antibodies were also calculated to show the impact of the HV loop redesign on the antibody accessibility of bnAb epitopes. The redesigned Env alleviated the clashes between 10-1074 and PGT135 and V1HV, but with limited effect on the clashes between CD4bs antibodies and V5HV. The greater depth and clash reduction for glycan-supersite epitope sites near V1HV was likely due to the fact that V1HV loops were longer than other HV loops and the HV loop redesign removed more spacer residues than for other HV loops (FIG. 1A, Table 1). The apex antibodies, PGT145 and VRC26.25, were not tested because their epitopes are quaternary and require modeling of the entire Env trimer, which was beyond the scope of the current endeavor.

[0176] The modeled structure of CRF01_AE V1HV was further examined because CRF01_AE Env has longer V1HV than those of subtypes B and C and shows high resistance to glycan supersite antibodies. The consensus sequence had a IGNITD (SEQ ID NO: 122) motif (147-152) as its V1HV C-anchor sites. AlphaFold2 predicted that I147 occupied the hydrophobic pocket formed by R327, K328, Y330, and P417, which corresponds to the spot targeted by F100Gon the CDR-H3 of 10-1074, as shown in FIG.2E. This meant that the 10-1074 epitope was hiddenAttorney Docket No.: HMJ-190-PCT with a median depth of 7.0 Å. After the HV loops redesign, the 24 amino acid-long V1HV loop (58.2 percentile in CRF01_AE) was replaced with a 9 amino acid-long loop (0.6 percentile in CRF01_AE, 01_AE.1). Hence, the median depth of 10-1074 epitope sites was reduced from 6.3 to 4.8 Å. The redesigned Env reduced the average number of clashes with 10-1074 from 120.8 to 9.2 atoms. See FIG. 2E. A similar decrease in the number of clashes was observed for another glycan supersite bnAb, PGT135, but the impact was limited for CD4bs antibodies (VRC01 and 3BNC117). The number of clashes with subtypes B, C, and CRF01_AE for antibodies 10-1074, PGT135, VRC01, and 3BBNC117 before and after redesign is shown below in Table 7A. Average decrease in clashes between antibody atoms and Env for unmodified and redesigned cognate HV loops (based on 100 conformations) is shown in Table 7B. Because the IGNITD (SEQ ID NO: 122) motif is highly conserved in CRF01_AE viruses, another version of V1HV (01_AE.2) was designed to preserve the IGNITD (SEQ ID NO: 122) motif, which may be important for CRF01_AE specific bnAbs. This variant CRF01_AE.2 design also improved antibody accessibility for glycan super site antibodies albeit to a lesser extent (Table 7B). Table 7A. Antibody atoms clashing with Env for unmodified and redesigned HV loops. HIV-1 10-1074 PGT135 VRC01 3BNC117 nredesigned cognate HV loops (based on 100 conformations). HIV-1 10-1074 clashes PGT135 clashes VRC01 clashes 3BNC117 clashes n

[0177] Without wishing to be bound by theory, it is postulated that the greater depth and clashAttorney Docket No.: HMJ-190-PCT reduction for glycan supersite epitope sites near V1HV was likely due to the fact that V1HV loops were longer than other HV loops, and the HV-loop redesign removed more spacer residues than for other HV loops. Example 4. Redesigned HV Loops Limit Interference of Glycan Supersite Epitope

[0178] To test if the HV loop redesign can promote bnAbs binding to Env, Env sequences were redesigned as sampled from one person living with HIV, 20337, who was enrolled in Kenya as part of the prospective acute HIV-1 infection RV217 cohort. Robb et al., N. Engl. J. Med., 2016, 374:2120-2130. The sequences sampled from this participant during acute HIV-1 infection showed that the infection was established with multiple HIV-1 founder variants and developed neutralization breadth a few years after diagnosis. Townsley 2021; and Lewitus et al., PLoS Pathog., 2022, 18:e1010369. Five Env variants that covered the diversity found in the first month of infection carried long V1HV loops (26 and 30 amino acids), while their glycan supersite epitopes were similar to those in known sensitive viruses in the CATNAP database. The V1HVs were redesigned to make them shorter (9 to 15 amino acids). The redesigned loops reduced the interference from V1HV on the epitopes of two glycan supersite bnAbs, 10-1074 and PGT135, as shown in Table 8 below and as illustrated in FIG. 4A and FIG. 4B. Table 8. Redesigned V1HV Loop of 5 Env Variants from 20337. Variant V1HV length (amino 10-1074 collisions PGT135 collisions n

[0179] The median depth of the 10-1074 epitope on five Env variants was reduced from 7.7- 8.3 Å to 5.8-7.0 Å (FIG. 4C), and for the PGT135 epitope, the median depth was reduced from 8.7-10.0 Å to 6.4-8.3 Å (FIG. 4D). The number of collisions was reduced from 73-101:Env interactions to 1-25 for 10-1074, and from 115-152 to 12-34 for PGT135:Env interactions.Attorney Docket No.: HMJ-190-PCT

[0180] The structural modeling disclosed herein uncovered a novel mechanism for CRF01_AE’s resistance to glycan supersite antibodies such as 10-1074. In addition to the absence of PNGS on site 332, I147 in CRF01_AE V1HV N-anchor sites was predicted to occupy the same location as the CDRH3 of 10-1074. In contrast, PGT128, a V3-glycan antibody whose epitope does not include I147 and is thus less occluded by the CRF01 AE V1HV, retained the capacity to neutralize half of the CRF01_AE viruses. It is possible that other viruses with long V1HV use a similar mechanism to obstruct the binding of glycan supersite bnAbs, highlighting that reducing the length of HV loops could play a key role in minimizing interference with bnAbs targeting adjacent epitopes. Example 5. Updated Env Consensus Sequences Are Functional Env Proteins

[0181] Pseudoviruses that expressed Env proteins for subtypes B, C, and CRF01_AE with either modified or unmodified loops were produced. It was shown that the seven Env tested were infectious. When normalized by the Gag-p24 quantities, viral titers were lower for the pseudoviruses with redesigned Env loops compared to those with unmodified loops for subtypes B and C, while it was the opposite for CRF01_AE (FIG. 5A).

[0182] Next, the consensus sequences were produced as gp140 glycoproteins. Negative-stain electron-microscopy (EM) showed that gp140s for subtypes B, C, and CRF01_AE were mostly open trimers and monomers, with only monomers being averageable from negative stain EM.

[0183] Finally, Env was stabilized in the prefusion conformation by combining previously reported modifications (Rutten et al., Cell Rep., 2018, 23:584–595; Rawi et al., 2020, Cell Rep., 33:108432) and three extra mutations. As stabilized trimers, produced by transient transfection and purified by GNA-lectin and size-exclusion chromatography, consensus B and C Env were just under 50% trimer by 2D averaging of negative-stain EM data (42 and 45%, respectively); the HV loop redesign marginally increased the population of trimer observed to 59 and 55%, respectively (FIG. 5B). To determine whether the HV loop redesign affected the opening of the trimer, 3D classification and refinement without any applied symmetry was used. For the subtype B consensus, 76% of trimers classified with the reconstruction identifiable as the closed prefusion state, while 24% appeared open, but still in a prefusion state (FIG. 5C). Results were similar for the HV loop redesign with 68% in the closed and 32% in the open prefusion state. In contrast, the subtype C consensus trimers were only at 31% in the closed prefusion state with 69% appearingAttorney Docket No.: HMJ-190-PCT open, with minimal changes with the HV loop redesign (29% in the closed and 71% in the open prefusion state). It cannot be ruled out that the conformational landscape of stabilized trimers is affected by the protein production and purification process as well as the adsorption to the carbon grid, which may explain the substantial fraction of monomer observed by negative-stain EM. Overall, these results suggest that the HV loop redesign marginally increased the trimer yield and had limited impact on the conformation of Env. Example 6. Improved Antibody Binding for Env-gp140s with Redesigned HV Loops

[0184] To test if the HV loop redesign improves antibody binding to Env, binding to the Env- gp140 glycoproteins was analyzed. Both versions of the consensus subtype B and C, plus the unmodified CRF01_AE consensus and the redesigned V1HV with the conserved IGNITD (SEQ ID NO: 122) motif (01_AE.2) were tested for binding to plasma pools and monoclonal antibodies with a bead-based multiplex immunoassay (Mdluli et al., PLoS Pathog., 2020, 16:e1009101; Merbah et al., J. Immunol. Methods, 2020, 487:112874). The pooled plasma samples represented 13 cohorts of people living with HIV-1 spanning diverse geographic areas and subtype / CRF distributions. Plasma antibodies across multiple clade-specific pools recognized the consensus B Env with redesigned HV loops at a slightly higher magnitude of binding compared to the Env with unmodified HV loops (FIG.6A). Similarly, monoclonal antibodies recognized the subtype B Env- gp140s with redesigned loops at a higher magnitude of binding, especially for antibodies targeting the CD4bs, V2 apex and glycan supersite epitopes (FIG. 6B). Summary measures for binding of pooled plasma or monoclonal antibodies demonstrated minor but significantly higher recognition of the subtype B Env-gp140s with redesigned HV loops when compared to the Env with unmodified loops (p ≤ 0.008) (FIG.6C-6D). A similar improvement of antibody binding was seen for the CRF01_AE consensus with redesigned HV loops (p = 0.008) (FIG. 6C-6D). The subtype C consensus proteins with or without redesigned loops showed no difference in antibody binding (FIG. 6C-6D), consistent with consensus C Env exhibiting high binding to plasma relative to the other subtype / CRF before the loops were redesigned. Binding to monoclonal antibodies that target epitopes that are not presented on gp140 such as MPER antibodies and PGDM1400 (which targets the quaternary V2-apex epitope) was low as expected and was excluded from the statistical test. The change in the predicted epitope depth was compared to the change in antibody binding signal and no significant correlation was found between them (Spearman’s ⍴ = 0.52, p-value = 0.15). InAttorney Docket No.: HMJ-190-PCT addition, 293 T cells were transfected with Env-gp160 plasmids corresponding to unmodified and redesigned HV loops and evaluated bnAb binding. For the two subtype B consensus sequences, comparable Env cell surface expression was shown, and staining with bnAbs such as 3BNC117 and PGDM1400 indicated that Env were expressed as closed trimers. Moreover, seven bnAbs tested showed increased binding to the Env with redesigned HV loops, suggesting improved epitope display.

[0185] Finally, how the Env consensus proteins (with or without modified HV loops) were neutralized by bnAbs was evaluated. A pseudovirus neutralization assay in TZM-bl cells was used and ten bnAbs targeting the CD4 binding site (VRC01 and 3BNC117), the V2-apex (PG9 and PGT145), the glycan supersite (10-1074 and PGT128), the MPER (10E8 and 4E10) and the carbohydrate-specific mannose-dependent (2G12) epitopes as well as the soluble CD4 (sCD4) were tested (FIG. 6E-6G for IC50 values and FIG. 6H-6J for IC80 values). When comparing the consensus Env with the redesigned HV loop Env to those with the native HV loops, the neutralization sensitivity did not increase or decrease consistently for all the bnAbs (FIG.6E, FIG. 6G, FIG. 6H, and FIG. 6J). When pooling data across the 10 bnAbs, there was no significant increase in neutralization sensitivity for subtype B and CRF01-AE with redesigned HV loops (one- tailed exact Wilcoxon-Pratt signed-rank test, p > 0.201) (FIG.6F). In contrast, the subtype C Env with redesigned HV loops was more sensitive to the ten bnAbs than the subtype C Env with unmodified loops (p = 0.047 for IC50 values (FIG. 6F) and p = 0.016 for IC80 values (FIG. 6I). One bnAb, PG9, showed a notable increase in sensitivity for the antigens with redesigned HV loops with a similar pattern across subtypes / CRF (FIG. 6G). Moreover, CRF01_AE consensus proteins with the redesigned HV loop version 1 became sensitive to the glycan supersite bnAb 10- 1074 (IC50 = 0.544 μg / ml) and to the sCD4 (IC50 = 0.617 μg / ml) while the original CRF01_AE consensus was resistant (IC50 / 80 > 50 μg / ml) (FIG. 6E, FIG. 6G, FIG. 6H, and FIG. 6J). Interestingly, the CRF01_AE consensus with redesigned HV loop version 1 was sensitive to 10- 1074 while the Env with version 2 of the redesign was resistant, in agreement with the antibody access predictions described above. The redesign version 1 is shorter in V1HV indicating that resistance to 10-1074 in CRF01_AE is associated with the V1 loop length and is not necessarily mediated by the lack of glycosylation at site 332 as neither redesigned consensus Env had a glycan at site 332.Attorney Docket No.: HMJ-190-PCT Example 7. In vitro Expression of HIV-1 Env Proteins in mRNA-Transfected Cells

[0186] 293T cells were transfected with 2 µg of mRNA encoding an HIV-1 env gene and harvested at day 2 post infection. Cells were split into separate staining reactions for monoclonal antibodies 3BNC117(BEI Resources, Cat. No. ARP-12474), 10-1074 (BEI Resources, Cat. No. ARP-12477), 10E8 (BEI Resources, Cat. No. ARP-12294), 447-52D (BEI Resources, Cat. No. ARP-4030), B12 (Polymun, Cat. No. AB011), PGDM1400 (Sok et al., Proc. Natl. Acad. Sci. U. S. A., 2014, 111(49):17624-17629), PGT151 (Falkowska et al., Immunity, 2014, 40(5):657-668), and 2G12 (produced in Expi293F cells as described above). Cells were washed and stained with anti-human IgG-PE at 1:250 dilution (SouthernBiotech, Cat. No. 2040-09) for 15 minutes and fixed in 0.5% paraformaldehyde. Fluorescence was measured on a FACSymphony A5 SE Cell Analyzer and analyzed in FlowJo v.9.9.6.

[0187] As shown in FIG. 7A-7B, there were robust Env protein expression from all subtype B consensus (B.con; FIG. 7A) and subtype B (B036; FIG. 7B) multiple founder virus variant (MFV) mRNAs on the cell surface. As also shown in FIG.7A-7B, some MFV variants displayed bNAb epitopes better than others (3BNC117 and B12: CD4 binding bNAbs; PGDM1400: Env trimer-specific bNAb; 10E8: MPER-specific bNAb; 10-1074: V-3 glycan-specific bNAb; 447- 52D: V-3 peptide-specific bNAb; PGT151: gp120-gp41 interphase-specific bNAb; 2G12: glycan- specific bNAb). Overall, subtype B MFV antigens were well expressed and displayed bNAb epitopes likely to engage B cells for developing broadly neutralizing antibody response in vivo. Example 8. LNP-mRNA Animal Immunization Studies

[0188] A total of three immunization studies have been conducted to date. Two of them were performed in mice and one was performed in rabbits. The immunogens were subtype B consensus (B.con) or subtype B (B036) multiple founder virus mRNAs encapsulated in lipid nanoparticles (LNP). The constructs tested in this study are summarized in Table 9. Table 9. Summary of Constructs Used in the LNP-mRNA Animal Immunization Studies. Construct Name Original Sequence Name SEQ ID NO:Attorney Docket No.: HMJ-190-PCT B.con.m.hv.4 B_2010s_hv_redesigned.O1.t937A.RnS.membrane 161 B.con.m.hv.5 B 2010s hv redesigned.O1.t889A.RnS.membrane 164.

[0189] The experimental design (see Tables 10A-10B) consisted of 6-week-old female BALB / c mice immunized through the intramuscular route with either 1 µg or 10 µg of LNP- mRNA on weeks 0, 2, and 4. In the first experiment, there were 14 groups, and each group had 5 mice / group. Mice were bled before immunization and then every two weeks until week 20 at which point the study was terminated. Blood was collected pre- and post-immunization until week 20. At week 20, the mice were euthanized, blood and immune organs (spleen, and bone marrow) were collected. Single cell suspensions of spleen and bone marrow cells were prepared and cryopreserved in liquid nitrogen. Serum was prepared from the clotted blood and aliquoted and stored at -80°C. The sera from the mouse studies were analyzed for binding antibodies by ELISA. Table 10A. Experimental Design of LNP-mRNA Mice Immunization Study 1. Mouse IDose / Mouse Grou DsLNP-mRNAAttorney Docket No.: HMJ-190-PCT 9 241-245 B036.m.3 10. . Mouse IDsDose / Mouse Group(5 mice / group)LNP-mRNA(µg mRNA)

[0190] Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Walter Reed Army Institute of Research [Assurance number D16-00596 (A4117-01)]. Blood collection was performed as per WRAIR / NMRC VSP Guidelines on blood collection. Mice were euthanized using CO2administered in a CO2chamber, via regulated flow valve (in accordance with the most current 2020 AVMA Guidelines on Euthanasia), followed by cervical dislocation at the various time points mentioned in the study to collect spleen, and bone marrow. Following euthanasia, mice were exsanguinated by cardiac puncture. Samples were processed as described above.

[0191] Vaccine Formulation: LNP-mRNA vaccines were prepared by diluting appropriate amounts of LNP-mRNA with PBS. The LNP-mRNA and the PBS were provided by Genevant Sciences Corporation. mRNAs were prepared and provided by University of Pennsylvania to Genevant and Genevant encapsulated the mRNA in LNPs. The working area and the pipettes usedAttorney Docket No.: HMJ-190-PCT were treated with RNAse Away prior to use and RNAse-free tips and tubes were used to ensure that there was no RNAse contamination, otherwise the RNA would be degraded. On the day of vaccination, 1 vial of LNP-mRNA was removed from the -80°C freezer and thawed at room temperature and then diluted with RNAse-free PBS. The prepared formulations were stored at 2- 8°C and no higher than 22°C until ready to be injected. The prepared vaccine formulations were injected into animals within 1 hour of preparation. Any remaining LNP-mRNA was discarded. Vaccine preparations were made for 6 mice to account for losses in the needle hub. Briefly, a vial containing 0.2 mL of LNP-mRNA was thawed as mentioned above. 30 µL of LNP-mRNA was mixed with 270 µL of RNAse-free PBS. Each mouse was administered with 50 µL of LNP-mRNA vaccine formulation containing 1 µg LNP-mRNA. To prepare 10 µg / mouse / dose, the vaccine formulation was calculated for 6 mice to account for losses in the needle hub. Briefly, a vial containing 0.5 mL of LNP-mRNA was thawed as mentioned above. Each mouse was administered 50 µL of LNP-mRNA vaccine formulation containing 10 µg LNP-mRNA. An example of the aliquots and the concentration is provided in Table 11. Table 11. An example of the aliquots and the concentration of the vaccine formulation. Quantity Sample Description mRNA Conc. Lot No.Attorney Docket No.: HMJ-190-PCT 3x2.0 mL mRNAlon High Binding U-bottom plates were coated with 100 µL of each of the HIV env proteins (1 µg / mL) in DPBS (pH 7.4), corresponding to the mRNA used for immunization and stored overnight at 4ºC. The plates were then blocked with 250 µL of blocking buffer, PBS containing 0.5% milk and 0.1% Tween 20 at room temperature (RT) for 2 hours. Individual serum samples were diluted in blocking buffer to an appropriate starting dilution and added to the first row of the 96-well plates in triplicates, where they were serially diluted two-fold in the blocking buffer in each row of the plates. The plates were then incubated at RT for 1 hour, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse secondary IgG (1:40,000 dilution in blocking buffer) and incubated at RT for 1 hour. Color was developed by the addition of peroxidase substrate components A and B and plates were incubated in the dark at RT for 1 hour. The reaction was stopped by addition of 100 µL per well of 1% sodium dodecyl sulfate, and the absorbance was measured within 30 minutes at 405 nm in a Spectramax M2 microplate reader (Molecular Devices). The results are expressed as endpoint titers defined as the reciprocal dilution that gives an absorbance value that is greater than or equal to twice the background values (antigen-coated wells that did not contain the test serum but had all the other components added). Antibody- positive (anti-subtype B gp120 seropositive samples from other studies) and antibody-negative controls were included on each plate.

[0193] The antigen-specific IgG endpoint titers for each group for each of the time points at which blood was collected are shown in FIG. 8A-8B. The data are plotted as bar graphs. Each bar represents the arithmetic mean + SEM of the end point titers of individual serum samples (N = 5), analyzed in triplicate. The peak antibody response at week 8 (4 weeks after the last boost; third immunization) was plotted for each of the groups for the 1 µg dose and 10 µg dose (FIG. 9A-9B). The mouse ELISA data showed that all the LNP-mRNA constructs tested expressed the protein in vivo and induced antibody responses. The antibody titers were maintained at 4 months post last immunization. In the case of subtype B consensus LNP-mRNAs, the lowest end point antibody titers were obtained with LNP-B.con.m.hv3 (pHIV16) MDV2 mRNA at both 1 µg and 10 µg doses (FIG. 8A and FIG. 9A). Based on this observation and the robust results obtained with the other constructs, it was decided to move ahead with a cocktail of either 3 or 5 LNP-mRNAAttorney Docket No.: HMJ-190-PCT constructs in rabbits in addition to testing singly the seed LNP-mRNA for the consensus and B036. For the cocktail containing the 3 LNP-mRNA constructs, LNP-B.con.m.hv3 (pHIV16) MDV2 was not used but the seed (B.con.m.hv1), B.con.m.hv2 (MDV3), and B.con.m.hv4, (MDV4) were used, while all 5 constructs (the seed and the 4 MFV constructs) were used in the 5-cocktail group. The amount of LNP-mRNA for the single and the 3 or 5 LNP-mRNA cocktails administered was a total of 20 µg / rabbit / dose. For the formulations that contained 3 LNP-mRNAs, the dose for each of the individual LNP-mRNA was 6.66 µg for a total of 20 µg and for the formulations that contained 5 LNP-mRNAs, the dose for each of the individual LNP-mRNA was 4 µg for a total of 20 µg.

[0194] Statistical Analyses: Statistical comparison between multiple groups in FIG. 8A-8B and FIG. 9A-9B was performed using ordinary one-way ANOVA with Tukey’s multiple comparison test. The significant p values are depicted in each of FIG. 8A-8B and FIG. 9A-9B. ii. Rabbit Immunization Studies

[0195] Female New Zealand white rabbits (19-week-old) were immunized through the intramuscular route with 20 µg of LNP-mRNA (Table 12). Each group of rabbits (n = 6 / group) was immunized with either the seed LNP-mRNA for B consensus or B036, or a cocktail of 3 or 5 LNP-mRNA on weeks 0, 4, and 8. Rabbits were bled pre- and post-immunization and then every two weeks until week 12 at which point the study was terminated. Blood was collected pre- and post-immunization until week 12. Serum was prepared from the clotted blood and aliquoted and stored at -80°C. The sera from the rabbit study were analyzed for binding antibodies by Luminex and for neutralizing antibodies. Table 12. Experimental Design of LNP-mRNA Rabbit Immunization Study. No. of D / R bbit)Attorney Docket No.: HMJ-190-PCT B036 465, 11, 12, 20,(5 formulations) 20

[0096] Multiplex Luminex Assay: e sera co ec ed rom e rabb s were ested simultaneously with a panel of antigens, including cognate MFVs (e.g., MFV_B036_seed, MFV_B2010sHVrd-3mer), gp70 scaffolded domains (e.g., gp70 V1 / V2 A244, gp70 V3 A244), consensus Envs (e.g., gp140 Con.A1_2010s_new_hv, gp140 Con.B_2010s_new_hv), additional Envs (e.g., gp120 Env, gp120 Con6), and controls (e.g., Mul V Trimer, gp70 Mul V, anti-IgG). Representative results obtained with eight gp140 Env antigens are shown in FIG. 10, which represent heterologous Env corresponding to the predominant HIV-1 clades. The corresponding antigens are listed on the top of each graph in FIG. 10 (Gp140 ConB: Env gp140 consensus for subtype B; Gp140 ConAE: Env gp140 consensus for CRF01_AE; Gp140 ConA: Env gp140 consensus for subtype A; Gp140 ConC: Env gp140 consensus for subtype C; Gp140 ConD: Env gp140 consensus for subtype D; Gp140 ConG: Env gp140 consensus for subtype G; Gp140 ConAG: Env gp140 consensus for CRF02_AG; and Gp140 ConM: Env gp140 consensus for HIV- 1 group M).

[0197] Overall, broad IgG responses were observed across clades for all groups of vaccinated animals, but multi-construct vaccinated animals appeared to have improved IgG binding antibody responses against cognate and heterologous antigens versus single-construct vaccinated animals. Moreover, it was observed that constructs based on subtype B consensus (B.con) elicited higher binding responses than constructs based on naturally occurring subtype B (B.036). Constructs based on subtype B consensus (B.con) also elicited detectable IgM response to the homologous antigen.Attorney Docket No.: HMJ-190-PCT

[0198] Neutralization Assessment: The sera collected from the rabbits were also analyzed for their neutralization breadth. Neutralization was measured using pseudoviruses expressing HIV-1 Env in a TZM-bl neutralization assay with 7-point titrations. Pseudoviruses expressed the two autologous Env and five heterologous Env, which corresponded to tier 1 pseudoviruses. Plasma samples were tested at 0, 6, 10, and 12 weeks.

[0199] The results obtained from this neutralization assessment are summarized in the heatmap shown in FIG. 11. The heatmap represents the neutralization titers measured against each pseudovirus (columns) in each animal at each time point (rows). Data are shown for the 8 rabbits in a group at a specific visit.

Claims

Attorney Docket No.: HMJ-190-PCT What is claimed:

1. A modified HIV-1 Envelope (Env) polypeptide comprising an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 116, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from N129D, T131S, D132N, G135D, G135E, G135D, S136L, S136Q, G137K, E143G, D175A, D175N, D267E, D267N, N268S, N268T A269F, A269G, N319D, N319T, R322D, R322Q, T323D, K324N, K324E, N326D, N326G, Q349K, V358I, M359I, Q375N, T380S, T380I, W381G, W381F, G385N, S386T, S390T, R438K, R438I, R438V, R438G, G440D, G440K, G441A, G441C, G441E, N442S, N443A, N443R, N443K, T446Q, T446K, E447G, T448N, G452Q, D455N, D455E, I496V, D655Q, G818T, G818L, L822I, L832S, and L837I; b) one or more repair modifications selected from S136T, S136N, E143D, A269T, N326E, T380S, G441A, G444N, T445S, T446N, and D655S; c) one or more trimer stabilization modifications selected from N290M, R292V, T306L, I516N, L537P, K569E, D570V, N632F, K636I, Q639V, A482C, T586C, and I540P; and / or c) a truncation after amino acid position 645 or amino acid position 693, or an immunogenic fragment or variant thereof.

2. The modified HIV-1 Env polypeptide of claim 2, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, or SEQ ID NO: 164, or an immunogenic fragment or variant thereof.

3. The modified HIV-1 Env polypeptide of claim 1 or 2, comprising the amino acid sequence of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, or SEQ ID NO: 164, or an immunogenic fragment or variant thereof.Attorney Docket No.: HMJ-190-PCT 4. A modified HIV-1 Envelope (Env) polypeptide comprising an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 108, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from V84Q, N129K, N134R, M141I, K162D, N177K, K217R, E283K, Q303E, T304S, T304M, A307E, K329D, F349T, N350K, G354A, S363N, S392K, V414A, G443E, N444G, N445L, T448E, D457E, R540T, S619Q, D657N, A764S, I819M, C827Y, R831K, F841N, A844N, A844S, and Q846P; b) one or more repair modifications selected from K162Q, S392T T304A, and G443A; c) one or more trimer stabilization modifications selected from N292M, R294V, T308L, I518N, L539P, K571E, D572V, N634F, K638I, K641V, A484C, T588C, and I542P; and / or d) a truncation after amino acid position 647 or amino acid position 695, or an immunogenic fragment or variant thereof.

5. The modified HIV-1 Env polypeptide of claim 4, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO: 178, or an immunogenic fragment or variant thereof.

6. The modified HIV-1 Env polypeptide of claim 4 or 5, comprising the amino acid sequence of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO: 178, or an immunogenic fragment or variant thereof.

7. A modified HIV-1 Envelope (Env) polypeptide comprising an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 105, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from H84N, N129S,Attorney Docket No.: HMJ-190-PCT N134K, G137N, N138T, S140E, G145S, E150K, K168P, N183S, K223S, N275G, N276S, A277V, G308S, E327D, N329I, T331N, K332Q, K332S, A341N, I354V, Q356T, P357L, M367I, K383R, N390S, G393K, S398G, G399N, G399E, G402R, V420D, R446A, G448S, G449E, G449N, N450S, N451G, N454K, N463F, T488S, R489S, R546K, E609R, N613G, N629D, N629S, D663E, R718S, G788E, L847I, and L852F; b) one or more repair modifications selected from S140T, E150D, K168Q, T331S, A341V, P357S, M367T, S398T, G449A, N451T, N463D, R489K, and E609D; c) one or more trimer stabilization modifications selected from N298M, R300V, T314L, I524N, L545P, K577E, D578V, N640F, K644I, K647V, A490C, T594C, and I548P; and / or d) a truncation after amino acid position 653 or amino acid position 701, or an immunogenic fragment or variant thereof.

8. The modified HIV-1 Env polypeptide of claim 7, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192, or an immunogenic fragment or variant thereof.

9. The modified HIV-1 Env polypeptide of claim 7 or 8, comprising the amino acid sequence of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, or SEQ ID NO: 192, or an immunogenic fragment or variant thereof.

10. A modified HIV-1 Envelope (Env) polypeptide comprising an amino acid sequence having one or more amino acid modifications as compared to the amino acid sequence of SEQ ID NO: 193, wherein the one or more amino acid modifications comprise: a) one or more multiple founder variant modifications selected from R320K, I365L, I366T, G372N, T381S, R460W, L457N, R460T, R460G, R460S, G462H, G463D, G463V, G463A, N464I, N464E, S466I, S466T, S467N, S467A, N469S, N469G, G475K, N478D, N478S,Attorney Docket No.: HMJ-190-PCT G644K, G644E, N678G, N678Y, R748Y, and N813D; b) one or more repair modifications selected from L81Q, Y92F, D129N, T163S, S168T, G172D, H184L, V330I, I434V, G463A, G475A, N478D, A538S, I587V, G623L, N678S, and N678T; c) one or more trimer stabilization modifications selected from N309M, K311V, T327L, T539N, L560P, R592E, D593V, T655F, K659I, Q662V, A505C, P609C, and I563P; and / or d) a truncation after amino acid position 668 or amino acid position 716, or an immunogenic fragment or variant thereof.

11. The modified HIV-1 Env polypeptide of claim 10, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, or an immunogenic fragment or variant thereof.

12. The modified HIV-1 Env polypeptide of claim 10 or 11, comprising the amino acid sequence of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, or an immunogenic fragment or variant thereof.

13. An artificial nucleic acid encoding the modified HIV-1 Env polypeptide of any one of claims 1-12.

14. The artificial nucleic acid of claim 13, wherein the nucleic acid is a messenger RNA (mRNA) molecule or a DNA molecule.

15. A vector comprising the artificial nucleic acid of claim 13 or 14.

16. A host cell comprising the vector of claim 15.Attorney Docket No.: HMJ-190-PCT 17. A composition comprising the modified HIV-1 Env polypeptide of any one of claims 1- 12, the artificial nucleic acid of claim 13 or 14, or the vector of claim 15.

18. The composition of claim 17, comprising: a) at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or variant thereof; b) at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or variant thereof; c) at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or variant thereof; or d) at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or variant thereof.

19. The composition of claim 17, comprising:Attorney Docket No.: HMJ-190-PCT a) artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, and SEQ ID NO: 164, or an immunogenic fragment or variant thereof; b) artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, or an immunogenic fragment or variant thereof; c) artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192, or an immunogenic fragment or variant thereof; or d) artificial nucleic acids encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or at least 14 different modified HIV-1 Env polypeptides selected from SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207, or an immunogenic fragment or variant thereof.

20. The composition of any one of claims 17-19, wherein the composition is an immunogenic composition.

21. A vaccine comprising the composition of claim 20, and a pharmaceutically acceptable carrier.Attorney Docket No.: HMJ-190-PCT 22. The vaccine of claim 21, further comprising an adjuvant.

23. The vaccine of claim 21 or 22, wherein the vaccine is an mRNA vaccine comprising at least one mRNA encoding the modified HIV-1 Env polypeptide.

24. A method of immunizing a subject, the method comprising administering to the subject in need thereof the vaccine of any one of claims 21-23.

25. The method of claim 24, wherein the method prevents HIV-1 infection in the subject, decreases the subject’s likelihood of getting HIV-1 infection, or reduces the subject’s likelihood of getting serious illness from HIV-1 infection.

26. The method of claim 24 or 25, wherein the subject is a human.

27. The method of any one of claims 24-26, wherein the vaccine is administered intramuscularly, intradermally, subcutaneously, intravenously, intranasally, by inhalation, or intraperitoneally.

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