WO2025072299A1 - Compositions comprising hiv envelopes to induce hiv-1 antibodies - Google Patents

Abstract

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

Description

COMPOSITIONS COMPRISING HIV ENVELOPES TO INDUCE

HIV-1 ANTIBODIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/585,199, filed on September 25, 2023, the entire contents of which are incorporated herein by reference.

GOVERNMENT INTERESTS

[0002] This invention was made with government support from the NIH, NIAID, Division of AIDS for UM1 grant AI144371. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirely⁷. Said ASCII copy, created on September 24, 2024, is named DU8322PCT Seq Listing. xml and is 21,974 bytes in size.

TECHNICAL FIELD

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

BACKGROUND

[0005] The development of a safe and effective HIV-1 vaccine is one of the highest priorities of the scientific community⁷ working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients, ART is not routinely available in developing countries.

[0006] One major target on the HIV Env for broadly neutralizing antibodies (bnAbs) can be the V3-glycan site. The N332 V3-gly can-dependent antibody lineage, DH270, was isolated from an individual living with HIV-1 called CH848 (Bonsignori et al. Sci Transl Med. 2017; 9(381)). The most potent and broad neutralizing antibody (DH270.6) of the lineage neutralized 55% of 208 viruses tested (see Bonsignori et al. supra).

SUMMARY OF THE INVENTION

[0007] Antibody improbable mutations R98T, L48Y, S27Y, and G57R were identified as sufficient for DH270 HIV-1 neutralization breadth. These amino acids are the target for selection by a vaccine aiming to elicit DH270-like antibodies. In some embodiments, one or more of these mutations are present in a recombinant HIV-1 envelope or peptide disclosed herein. The recombinant HIV-1 envelopes can be used to induce an immune response in a subject. In some embodiments, these mutations are present in the context of SEQ ID NO: 1 or SEQ ID NO: 2, as disclosed herein. In some embodiments, these mutations are present in the context of other HIV-1 envelopes. In some embodiments, these other HIV-1 envelopes can be 90, 91, 92, 93, 94, 95, 96. 97. 98 or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2. [0008] In certain embodiments, the invention provides compositions and methods for induction of an immune response, for example cross-reactive (broadly) neutralizing (bn) Ab induction.

[0009] In certain aspects the invention provides a recombinant protein encoding a recombinant protein comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 1 (HV1303395 JRFL SOSIPv6_MCD5_101nQQavi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 2 (HV 1303396 JRFL SOSIPv6_MCD5_cSorta). In certain aspects the invention provides a recombinant nucleic acid encoding a recombinant protein comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 3 (HV1303395 JRFL SOSIPv6_MCD5_101nQQavi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 4 (HV1303396 JRFL SOSIPv6 MCD5 cSorta).

[0010] In certain aspects the invention provides a selection of HIV- 1 envelopes for use as prime and boost immunogens in methods to induce HIV-1 neutralizing antibodies. In certain aspects, the invention provides a selection of HIV-1 envelopes for use as a boost immunogen in methods to induce HIV-1 neutralizing antibodies.

[0011] In certain aspects the invention provides a selection of a series of immunogens and immunogen designs for induction of neutralizing HIV-1 antibodies, e.g. but not limited to V3 glycan epitope targeting antibodies. In some embodiments, the selection comprises an immunogen comprising HV1303395 JRFL SOSIPv6_MCD5_l OlnQQavi or HV1303396 JRFL SOSIPv6_MCD5_cSorta.

[0012] In certain embodiments, the methods use compositions comprising HIV-1 envelope immunogens designed to bind to precursors, and/or unmutated common ancestors (UCAs) of different HIV-1 bnAbs. In certain embodiments, these are UCAs of V1V2 glycan and V3 glycan binding antibodies. Thus, in certain embodiments the invention provides HIV-1 envelope immunogen designs with multimerization and variable region sequence optimization for enhanced UCA-targeting. In certain embodiments the invention provides HIV-1 envelope immunogen designs with multimerization and variable region sequence optimization for enhanced targeting and inductions of multiple antibody lineages, e.g. but not limited to V3 lineage, V1V2 lineages of antibodies.

[0013] In certain aspects the invention provides compositions comprising a selection of HIV-1 envelopes and/or nucleic acids encoding these envelopes as described herein, for example, but not limited to designs as described herein. Without limitations, these selected combinations comprise envelopes which provide representation of the sequence (genetic) and antigenic diversity of the HIV-1 envelope variants which lead to the induction of V1V2 glycan and V3 glycan antibody lineages.

[0014] In certain aspects the invention provides compositions comprising recombinant HIV-1 envelopes and/or nucleic acids encoding these envelopes with modifications to the VI loop at positions 134-138 (HXB2 numbering). In some embodiments, the modification is HEKGG. Such a modification can be incorporated into any HIV-1 envelope sequences from the CH848 infected individual and variants thereof. See e.g., US2020/0113997 incorporated herein by reference in its entirety including Figures 40A-C, 41 A-41 C, 44A-D, 45, 46, 47A, 49A-B, 50A-D, 51, 52A-B, 53A, 53D, 54A-F, 77A-L, and 78A-B and SEQ ID NOs disclosed therein. In some embodiments, such a modification can be incorporated into envelope JR-FL. In some embodiments, such a modification can be incorporated into envelope CH848.3.D0949.10.17 (also referred to as CH848.d0949. 10.17WT) and variants thereof, including, but not limited to, CH848.d0949. 10. 17 DT (also referred to as CH848.d0949.10.17.N133D.N138T). In some embodiments, such a modification can be incorporated into envelope CH848.d0808. 15. 15 and variants thereof. In some embodiments, such a modification can be incorporated into envelope CH848.d0358.80.06 and variants thereof. In some embodiments, such a modification can be incorporated into envelope CH848.dl432.5.41 and variants thereof. In some embodiments, such a modification can be incorporated into envelope CH848.dl621.4.44 and variants thereof. In some embodiments, such a modification can be incorporated into envelope CH848.dl305.10.35 and variants thereof. In some embodiments, such a modification can be incorporated into envelope CH848.0358.80.06. In some embodiments, such a modification can be incorporated into envelope CH848.1432.5.41. In certain embodiments, the invention provides compositions comprising recombinant HIV- 1 envelope HV1302295 JRFL SOSIPv6_MCD5_101nQQavi or HV 1303396 JRFL SOSIPv6_MCD5_cSorta and/or nucleic acids encoding these envelopes. [0015] In some embodiments, the recombinant HIV-1 envelope comprises a tag at the C- terminal of the envelope. In some embodiments, the tag is Avi tag. In some embodiments, the tag is sortase A tag. In some embodiments, the recombinant HIV-1 envelope further comprises a tag and a linker.

[0016] In certain aspects the recombinant HIV-1 envelope optionally comprises any combinations of additional modifications, such as the modifications described in Table 2. In certain aspects the invention provides a recombinant HIV-1 envelope comprising a shortened VI region (e.g., 17 amino acid (17aa) or shorter VI region), lacking glycosylation at position N133 and N138 (HXB2 numbering), comprising glycosylation at N301 (HXB2 numbering) and N332 (HXB2 numbering), comprising modifications wherein glycan holes are filled (e.g, at positions 230, 241, 289, or other sites identified to be a glycosylation site in more than 80% of HIV-1 envelope sequences (HXB2 numbering)), comprising the “GDIR” (SEQ ID NO: 5) or "GDIK " (SEQ ID NO: 6) motif, or any trimer stabilization modifications, UCA targeting modification, immunogenicity modification, or combinations thereof, for example but not limited to these described in Table 2. In certain embodiments, the modifications where glycan holes are filled comprise D230N, H289N, and P291S. In some embodiments, the glycan hole in envelope JR-FL at HXB2 position 241 is filled. In certain embodiments the recombinant envelope optionally comprises any combinations of these modifications.

[0017] In certain embodiments, the inventive designs comprise glycan holes filled with the introduction of new glycosylation sites to prevent the binding of strain-specific antibodies that could hinder broad neutralizing antibody development (Wagh, Kshitij et al.

“Completeness of HIV-1 Envelope Glycan Shield at Transmission Determines Neutralization Breadth.’⁷ Cell reports vol. 25.4 (2018): 893-908.e7. doi: 10.1016/j.celrep.2018.09.087;

Crooks, Ema T et al. “Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Gly can-Deficient Patches Proximal to the CD4 Binding Site.” PLoS pathogens vol. 11,5 el004932. 29 May. 2015, doi: 10.1371/joumal.ppat. l004932).

[0018] In certain embodiments, the recombinant HIV-1 envelope comprises any envelope sequence from the CH848 infected individual and variants thereof comprising the modification to the VI loop described herein. Examples of CH848 envelopes are described in Table 2. In some embodiments, the recombinant HIV-1 envelope comprises any envelope sequence from the CH848 infected individual and variants thereof comprising HEKGG at positions 134-138 (HXB2 numbering).

[0019] In certain embodiments the envelope is a protomer which could be comprised in a stable trimer.

[0020] In certain embodiments the envelope comprises additional mutations stabilizing the envelope trimer. In certain embodiments these include, but are not limited to, SOSIP mutations. In certain embodiments mutations are selected from sets F1-F14, VT1-VT8 mutations described herein, or any combination or subcombination within a set. In certain embodiments, the selected mutations are F14. In other embodiments, the selected mutations are VT8. In certain embodiments, the selected mutations are F 14 and VT8 combined.

[0021] In certain embodiments, the invention provides a recombinant HIV-1 envelope of Table 1. In certain embodiments, the invention provides a recombinant HIV-1 envelope of Figure 7. In certain embodiments, the invention provides a nucleic acid encoding any of the recombinant envelopes. In certain embodiments, the nucleic acids comprise an mRNA formulated for use as a pharmaceutical composition.

[0022] In certain embodiments, the inventive designs comprise specific changes to the V 1 loop at positions 134-138, as shown in Figure 7. In certain embodiments, JR-FL envelope comprises HEKGG at positions 134-138 and is referred to as JRFL SOSIPv6_MCD5_101nQQavi. In certain embodiments. JR-FL envelope comprises HEKGG at positions 134-138 and is referred to JRFL SOSIPv6_MCD5_cSorta.

[0023] In non-limiting embodiments, the envelope in the selections for immunization are included as trimers, protein and/or mRNA. In non-limiting embodiments, the envelope in the selections for immunization are included as nanoparticles, protein and/or mRNA. The designation scNP refers to a non-limiting embodiment of a protein nanoparticle formed by sortase conjugation reaction. In non-limiting embodiments, nanoparticles comprise fusion proteins, for example ferritin-envelope fusion proteins. [0024] In certain embodiments, the inventive designs comprise modifications, including without limitation fusion of the HIV-1 envelope with ferritin using linkers between the HIV-1 envelope and ferritin designed to optimize ferritin nanoparticle assembly.

[0025] In certain embodiments, the invention provides HIV-1 envelopes comprising Lys327 (HXB2 numbering) optimized for administration as a prime to initiate V3 glycan antibody lineage, e.g. DH270 antibody lineage.

[0026] In certain embodiments, the invention provides HIV-1 envelopes comprising Lysl69 (HXB2 numbering).

[0027] In certain embodiments, the invention provides a composition comprising any one of the inventive envelopes, e.g., as disclosed in Table 1, or nucleic acid sequences encoding the same. In certain embodiments, the nucleic acid is mRNA. In certain embodiments, the mRNA is comprised in a lipid nano-particle (LNP).

[0028] In certain embodiments, the invention provides compositions comprising a nanoparticle which comprises any one of the envelopes of the invention, e.g., as disclosed in Table 1

[0029] In certain embodiments, the invention provides compositions comprising a nanoparticle which comprises any one of the envelopes of the invention, e.g.. as disclosed in Table 1, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

[0030] In certain aspects, the invention provides a composition comprising a nanoparticle and a carrier, w herein the nanoparticle comprises trimers of any of the recombinant HIV-1 envelopes, e.g. as disclosed in Table 1. In certain embodiments, the nanoparticle is a ferritin self-assembling nanoparticle. In certain embodiments, the nanoparticle comprises multimers of trimers. Provided also are method for using these compositions comprising nanoparticles. [0031] In certain embodiments, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant HIV-1 envelopes of the invention e.g., as disclosed in Table 1. or compositions comprising these recombinant HIV-1 envelopes, in an amount sufficient to induce an immune response. In certain embodiments, the composition is administered as a prime and/or a boost. In certain embodiments, the composition is administered as a prime. In certain embodiments, the composition is administered as a boost. In certain embodiments, the composition comprises nanoparticles. In certain embodiments, methods of the invention further comprise administering an adjuvant. [0032] In certain embodiments, the invention provides a composition comprising a plurality of nanoparticles comprising a plurality of the recombinant HIV-1 envelopes or trimers of the invention, e.g., as disclosed in Table 1. In non-limiting embodiments, the envelopes/trimers of the invention are multimeric when comprised in a nanoparticle. The nanoparticle size is suitable for delivery. In non-liming embodiments the nanoparticles are ferritin-based nanoparticles.

[0033] In certain aspects, the invention provides nucleic acids comprising sequences encoding proteins of the invention, e.g., as disclosed in Table 1. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs, modified or unmodified, suitable for use any use, e.g., but not limited to use as pharmaceutical compositions. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0034] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive HIV-1 envelopes, e.g., as disclosed in Table 1. In certain embodiments, these are optionally formulated in lipid nanoparticles (LNPs). In certain embodiments, the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly -A tail, 5 ’cap.

[0035] In certain embodiments, the nucleic acids are formulated in lipid, such as but not limited to LNPs. Non-limiting embodiments include LNPs without polyethylene glycol.

[0036] In certain aspects the invention provides nucleic acids encoding the inventive protein designs. In non-limiting embodiments, the nucleic acids are mRNA, modified or unmodified, suitable for any use, e.g, but not limited to use as pharmaceutical compositions. In certain embodiments, the nucleic acids are formulated in lipid, such as but not limited to LNPs.

[0037] In certain aspects the invention provides a method of inducing an immune response comprising administering an immunogenic composition comprising a prime immunogen followed by at least one boost immunogen from Table 1, wherein the boost immunogens are administered in an amount sufficient to induce an immune response. In certain embodiments, the prime is one of the CH848.0949.10.17DT, CH848.0949. 10. 17Dte, CH848.d0949.10.17DT.GS, or CH848.d0949.10.17DT.GS comprising additional modifications D230N.H289N.P291S.E169K designs. See Table 2 and W02022/087031 which content is herein incorporated by reference in its entirety. In certain embodiments, the first boost is one of the CH848.0949.10. 17WT, CH848.0949. 10.17WTe designs. See Table 2 and W02022/087031 which content is herein incorporated by reference in its entirety. In certain embodiments, the first boost is one of the CH848.0949. 10. 17DT or

CH848.0949. 10. 17Dte designs. See Table 2. In certain embodiments, the first boost is one of the JRFL-MCD5. See Table 1. In certain embodiments, the boost is CH848.0358.80.06 or CH848.1432.5.41. In some embodiments, the modification to the VI loop described herein can be incorporated into the envelope used as the prime and/or boost. In some embodiments, the method further comprises administering an immunogenic composition comprising any HIV-1 envelope sequence from the CH848 infected individual and variants thereof comprising the modification to the VI loop described herein. In some embodiments, the method comprises administering an immunogenic composition comprising any HIV-1 envelope sequence from the CH848 infected individual and variants thereof comprising the modification to the VI loop described herein as a boost.

[0038] In certain embodiments, the methods further comprise administering a boost from Table 1, wherein the boost is JRFL-MCD5 in any suitable form.

[0039] In certain embodiments, the methods further comprise administering a boost from Table 1, wherein the boost is an envelope from Table 1 in any suitable form. In certain embodiments, the boost comprises envelope HV1303395 JRFL SOSIPv6_MCD5_101nQQavi, or HV1302296 JRFL SOSIPv6_MCD5_cSorta. In certain embodiments, the boost comprises any HIV-1 envelope comprising modifications to the VI loop at positions 134-138 (HXB2 numbering). In certain embodiments, the modification to the VI loop at positions 134-138 is HEKGG (SEQ ID NO: 7).

[0040] In certain embodiments, the prime and/or boost immunogen are administered as a nanoparticle. In certain embodiments, the nanoparticle is a ferritin nanoparticle. In certain embodiments, the methods further comprise administering the prime and/or boost immunogen as a mRNA-LNP formulation.

[0041] In certain embodiments, the methods further comprise administering any suitable adjuvant.

BRIEF DESCRIPTION OF DRAWINGS

[0042] The patent or application file contains at least one drawing executed in color. [0043] Figure 1 depicts a schematic of the overall vaccination strategy in knock-in mice to generate the 4 mutations of interest. [0044] Figure 2 is a heatmap showing the addition of the S27Y substitution to vaccine- induced mAh increases its neutralization breadth and potency.

[0045] Figure 3 shows an example design of an immunogen that is capable of selecting for the S27Y substitution in DH270 antibodies. Mammalian cell display of envelope variants was used to select for an envelope that binds with high affinity to the DH270 UCA antibody with S27Y but binds lowly to DH270 precursor antibody without the S27Y substitution.

[0046] Figure 4 shows example selection of HIV- 1 Env with high binding to DH270UCA3_S27Y mutant. Three rounds of sorting cells that bound to DH270 UCA+S27Y was performed. There was a clear binding population of cells after three rounds of sorting.

[0047] Figure 5 illustrates examples of selected JR-FL Env variant exhibiting significant binding to DH270UCA3_S27Y compared to the wildtype Env. The cell population expressing envelope in culture bound to DH270 UCA S27Y, whereas the starting wildtype JR-FL Env did not bind. The binding was specific as DH270 UCA+S27Y bound in a dosedependent manner to the selected cells,

[0048] Figure 6 shows examples of cell-surface expressed selected Env has improved binding to DH270 UCA with G57R and R98T improbable mutations. DH270 UCA with a G57R substitution added, bound to both the wildtype JR-FL Env displayed on the surface of cells, but bound better to the Env variant expressed by sorted cells. DH270 UCA+ R98T exhibited dose-dependent binding to the selected Env variant as well. A small effect was seen on L48Y or UCA binding.

[0049] Figure 7 shows example results of next generation sequencing (NGS) on sorted cells and resulted in identification of a ¹³⁴VNATN¹³⁸ (SEQ ID NO: 16) to ¹³⁴HEKGG¹³⁸ (SEQ ID NO: 7) mutation in the VI loop. Soluble Env trimer was expressed and binding was characterized.

[0050] Figure 8 shows example ELISA binding magnitude of JR-FL MCD5 binding to DH270 antibodies with the four functional mutations. Using the soluble JR-FL MCD5 envelope, the binding magnitude for DH270 antibodies to JR-FL MCD5 by ELISA and biolayer interferometry (BLI) was determined.

[0051] Figure 9 shows example results of kinetic BLI assay of JR-FL MCD5 and JR-FL binding to DH270 lineage. JR-FL MCD5 binds with higher apparent affinity to DH270 antibodies than wildtype JRFL. The DH270 antibody that was the target for MCD5 directed evolution is highlighted in yellow. The engineered envelope has a 38-fold increase in apparent binding affinity for the target antibody S27Y.

[0052] Figure 10 shows an example that MCD5 binds stronger than JR-FL to CH848 serum IgG during chronic infection.

[0053] Figure 11 shows example serum binding curves for MCD5 and JR-FL (A) and neutralization breadth (B). Binding to MCD5 increases when serum neutralization breadth increases.

[0054] Figure 12 shows an example that an increase in binding to CH848 serum IgG occurs when DH270 sequences are detected in peripheral B cells.

[0055] Figure 13 shows an example that JR-FL MCD5 Env binds to vaccinated mouse serum IgG.

[0056] Figure 14 shows example potential immunization regimens in mice with MCD5 Env.

[0057] Figure 15 shows example vaccination regimens performed in a HIV-1 broadly neutralizing antibody DH270 precursor knock-in mouse model. The immunization regimens compare JRFL MCD5 boosting immunization to CH848. 10. 17DT prime and CH848. 10. 17 boost.

[0058] Figure 16 shows an example of superior induction of autologous tier 2 neutralizing antibodies by JRFL MCD5 boost compared to CH848.10.17 boost. The bars represent the group geometric mean. N332T mutation in the DH270 epitope on the virus knocks down the serum neutralization indicating the antibodies are on target.

[0059] Figure 17 shows an example that JRFL.MCD5 boost elicits higher heterologous neutralizing antibodies than CH848. 10.17 boost. The heterologous neutralization of Q23. 17 is dependent on the DH270 epitope.

[0060] Figure 18 shows an example that MCD5 trimer immunization increases the S27Y substitution frequency in DH270 UCA sequences compared to CH848. 10. 17 boosting alone. MCD5 as the last 4 immunization increased the median frequency of S27Y substitution by one log.

[0061] Figure 19 shows an example that MCD5 Trimer immunization increases the combination of light chain mutations while not adversely affecting G57R+R98T frequency in DH270 heavy chain sequences. Together all 4 key substitutions are the key substitutions to select for in DH270 antibodies, thus MCD5 boosting shows high frequencies of all four mutations.

[0062] Figure 20 shows example vaccination regimens performed in a HIV-1 broadly neutralizing antibody DH270 precursor knock-in mouse model. The immunization regimens compare JRFL MCD5 boosting after priming with CH848. 10.17DT only in order to shorten the vaccination regimen.

[0063] Figure 21 shows four example MCD5 boosts that elicit comparable serum autologous neutralizing antibodies in DH270 UCA knock-in mice as eight 10.17 boosts. [0064] Figure 22 shows an example that boosting 4 times with MCD5 trimer generated more potent heterologous neutralization than boosting 8 times with CH848. 10. 17. Each symbol represents the group geometric mean for a single virus. Five viruses were tested in total.

[0065] Figure 23 shows an example that boosting four times with MCD5 trimer after priming elicited higher neutralizing antibodies than 10.17DT prime with 10.17 boosts. However, 10.17DT/MCD5 trimer regimen elicited lower neutralization titers than prime followed by four 10.17 boosts and four MCD5 boosts. Each symbol represents the group geometric mean for a single virus. Five viruses were tested in total.

[0066] Figure 24 shows example vaccination regimens performed in a HIV-1 broadly neutralizing antibody DH270 precursor knock-in mouse model. The immunization regimens compare four JRFL MCD5 sortase A conjugate nanoparticle boosts in place of 10.17 Env trimer boosting and MCD5 Env trimer boosting in order to shorten the vaccination regimen. [0067] Figure 25 shows an example that boosting four times with MCD5 scNP elicited more potent heterologous neutralization than MCD5 trimer boosts. Heterologous neutralization titer was comparable to group 2, but was four immunizations shorter.

[0068] Figure 26 shows an example that boosting with MCD5 scNP only selected S27Y to comparable frequencies as boosting with both 10. 17 Env trimer followed by MCD5 Env trimer. Symbols represent the frequency of DH270 sequences with S27Y substitution with the group median represented by the horizontal bar.

[0069] Figure 27 shows boosting with MCD5 selected DH270 antibodies with all 4 key mutations. Amino acid alignment of DH270 lineage precursor is shown on top for comparison. DH270 is a broadly neutralizing antibody from the human living with HIV-1. DH270.mul06 is the vaccine-elicited antibody. Enlarged boxes show the mouse antibody has all four key target mutations. This antibody was isolated from a mouse immunized with CH848. 10. 17DT prime followed by MCD5 Env Trimer.

DETAILED DESCRIPTION

[0070] The development of a safe, highly efficacious prophylactic HIV-1 vaccine is of paramount importance for the control and prevention of HIV- 1 infection. A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) (Immunol. Rev. 254: 225-244. 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not induced by current vaccines.

[0071] For the past 25 years, the HIV vaccine development field has used single or prime boost heterologous Envs as immunogens, but to date has not found a regimen to induce high levels of bnAbs.

[0072] Recently, a new paradigm for design of strategies for induction of broadly neutralizing antibodies was introduced, that of B cell lineage immunogen design (Nature Biotech. 30: 423, 2012) in which the induction of bnAb lineages is recreated. It was recently demonstrated the power of mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction (Nature 496: 469, 2013). The invention provides methods of using pan bnAb envelope immunogens.

[0073] In certain aspects, the invention provides compositions for immunizations to induce lineages of broad neutralizing antibodies. In certain embodiments, there is some variance in the immunization regimen; in some embodiments, the selection of HIV- 1 envelopes may be grouped in vanous combinations of primes and boosts, either as nucleic acids, proteins, or combinations thereof. In certain embodiments the compositions are pharmaceutical compositions which are immunogenic. In certain embodiments, the compositions comprise amounts of envelopes which are therapeutic and/or immunogenic. [0074] In one aspect the invention provides a composition for a prime boost immunization regimen comprising any one of the envelopes described herein, or any combination thereof wherein the envelope is a prime or boost immunogen. In certain embodiments the composition for a prime boost immunization regimen comprises one or more envelopes described herein.

[0075] In certain embodiments, the compositions contemplate nucleic acid, as DNA and/or RNA, or recombinant protein immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and/or RNA, immunization either alone or in combination with recombinant envelope protein(s).

[0076] In some embodiments the antigens are nucleic acids, including but not limited to mRNAs which could be modified and/or unmodified. See US Pub 20180028645A1. US Pub 20170369532, US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261172, US Pub 20150038558, US Pub 20160032316, US Pub 20170043037, US Pub 20170327842, each content is incorporated by reference in its entirety. mRNAs delivered in LNP formulations have advantages over non-LNPs formulations. See US Pub 20180028645A1.

[0077] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted into an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant. [0078] In certain embodiments the induced immune response includes induction of antibodies, including but not limited to autologous and/or cross-reactive (broadly) neutralizing antibodies against HIV-1 envelope. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are known in the art and are also described herein.

[0079] In certain aspects the invention provides an expression vector compnsing any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector.

[0080] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0081] The envelope used in the compositions and methods of the invention can be a gpl60, gpl50, gpl45, gpl40, gpl20, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. In certain embodiments the composition comprises envelopes as trimers. In certain embodiments, envelope proteins are multimerized, for example trimers are attached to a particle such that multiple copies of the trimer are attached and the multimerized envelope is prepared and formulated for immunization in a human. In certain embodiments, the compositions comprise envelopes, including but not limited to trimers as a particulate, high- density array on liposomes or other particles, for example but not limited to nanoparticles. In some embodiments, the trimers are in a well ordered, near native like or closed conformation. In some embodiments the trimer compositions comprise a homogenous mix of native like trimers. In some embodiments the trimer compositions comprise at least 85%, 90%, 95% native like trimers.

[0082] In certain embodiments the envelope is any of the forms of HIV-1 envelope. In certain embodiments the envelope is gpl20. gpl40. gpl45 (i.e.. with a transmembrane domain), or gpl50. In certain embodiments, gp!40 is designed to form a stable trimer. See Table 1 for non-limiting examples of sequence designs. In certain embodiments envelope protomers form a trimer which is not a SOSIP timer. In certain embodiment the trimer is a SOSIP based trimer wherein each protomer comprises additional modifications. In certain embodiments, envelope trimers are recombinantly produced. In certain embodiments, envelope trimers are purified from cellular recombinant fractions by antibody binding and reconstituted in lipid comprising formulations. See for example W02015/127108 titled “Trimeric HIV-1 envelopes and uses thereof’ and US2020/0002383 which content is herein incorporated by reference in its entirety. In certain embodiments the envelopes of the invention are engineered and comprise non-naturally occurring modifications.

[0083] In certain embodiments, the envelope is in a liposome. In certain embodiments the envelope comprises a transmembrane domain with a cytoplasmic tail, wherein the transmembrane domain is embedded in a liposome. In certain embodiments, the nucleic acid comprises a nucleic acid sequence which encodes a gpl20, gpl40, gpl45, gpl50, or gpl60. [0084] In certain embodiments, where the nucleic acids are operably linked to a promoter and inserted in a vector, the vector is any suitable vector. Non-limiting examples include, VSV, replicating rAdenovirus type 4, MV A, Chimp adenovirus vectors, pox vectors, and the like. In certain embodiments, the nucleic acids are administered in NanoTaxi block polymer nanospheres. In certain embodiments, the composition and methods comprise an adjuvant. Non-limiting examples include, 3M052, AS01 B, AS01 E, gla/SE, alum, Poly I poly C (poly IC), polylC/long chain (LC) TLR agonists, TLR7/8 and 9 agonists, or a combination of TLR7/8 and TLR9 agonists (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329-3339), or any other adjuvant. Non-limiting examples of TLR7/8 agonist include TLR7/8 ligands, Gardiquimod, Imiquimod and R848 (resiquimod). A non-limiting embodiment of a combination of TLR7/8 and TLR9 agonist comprises R848 and oCpG in STS (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329-3339). In nonlimiting embodiments, the adjuvant is an LNP. See e.g., without limitation Shirai et al. “Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses” Vaccines 2020, 8, 433; doi:10.3390/vaccines8030433, published 3 August 2020.

[0085] In non-limiting embodiments, LNPs used as adjuvants for proteins or mRNA compositions are composed of an ionizable lipid, cholesterol, lipid conjugated with polyethylene glycol, and a helper lipid. Non-limiting embodiments include LNPs without polyethylene glycol.

[0086] In certain aspects the invention provides a cell comprising a nucleic acid encoding any one of the envelopes of the invention suitable for recombinant expression. In certain aspects, the invention provides a clonally derived population of cells encoding any one of the envelopes of the invention suitable for recombinant expression. In certain aspects, the invention provides a stable pool of cells encoding any one of the envelopes of the invention suitable for recombinant expression.

[0087] In certain aspects, the invention provides a recombinant HIV-1 envelope polypeptide as described here, wherein the polypeptide is a non-naturally occurring protomer designed to form an envelope trimer. The invention also provides nucleic acids encoding these recombinant polypeptides. Non-limiting examples of amino acids and nucleic acid of such protomers are disclosed herein. [0088] In certain aspects the invention provides a recombinant trimer comprising three identical protomers of an envelope. In certain aspects the invention provides an immunogenic composition comprising the recombinant trimer and a carrier, wherein the trimer comprises three identical protomers of an HIV-1 envelope as described herein. In certain aspects the invention provides an immunogenic composition comprising nucleic acid encoding these recombinant HIV-1 envelope and a carrier.

[0089] Described herein are nucleic and amino acid sequences of HIV-1 envelopes. The sequences for use as immunogens are in any suitable form. In certain embodiments, the described HIV-1 envelope sequences are gpl60s. In certain embodiments, the described HIV-1 envelope sequences are gpl20s. Other sequences, for example but not limited to stable SOSIP trimer designs, gpl45s, gpl40s, both cleaved and uncleaved, gpl40 Envs with the deletion of the cleavage (C) site, fusion (F) and immunodominant (I) region in gp41— named as gpl40ACFI (gpl40CFI), gpl40 Envs with the deletion of only the cleavage (C) site and fusion (F) domain - named as gpl40ACF (gpl40CF), gpl40 Envs with the deletion of only the cleavage (C) — named gpl40AC (gpl40C) (See e.g. Liao et al. Virology 2006, 353, 268-282), gpl50s, gp41s, can be readily derived from the nucleic acid and amino acid gpl60 sequences. In certain embodiments the nucleic acid sequences are codon optimized for optimal expression in a host cell, for example a mammalian cell, a rBCG cell or any other suitable expression system.

[0090] An HIV-1 envelope has various structurally defined fragments/forms: gpl60; gp 140— including cleaved gpl40 and uncleaved gpl40 (gpl40C), gpl40CF, or gpl40CFI; gpl20 and gp41. A skilled artisan appreciates that these fragments/forms are defined not necessarily by their crystal structure, but by their design and bounds within the full length of the gpl60 envelope. While the specific consecutive amino acid sequences of envelopes from different strains are different, the bounds and design of these forms are well known and characterized in the art.

[0091] For example, it is well known in the art that during its transport to the cell surface, the gpl60 polypeptide is processed and proteolytically cleaved to gpl20 and gp41 proteins. Cleavages of gpl60 to gpl20 and gp41 occurs at a conserved cleavage site “REKR.’" (SEQ ID NO: 8) See Chakrabarti et al. Journal of Virology’ vol. 76, pp. 5357-5368 (2002); see, e.g., Figure 1. and second paragraph in the Introduction on p. 5357; Binley et al. Journal of Virology vol. 76, pp. 2606-2616 (2002) for example at Abstract; Gao et al. Journal of Virology vol. 79, pp. 1154-1163 (2005); Liao et al. Virology vol. 353(2): 268-282 (2006). [0092] The role of the furin cleavage site was well understood both in terms of improving cleavage efficiency, see Binley et al. supra, and eliminating cleavage, see Bosch and Pawlita, Virology 64 (5):2337-2344 (1990); Guo et al. Virology 174: 217-224 (1990); McCune et al. Cell 53:55-67 (1988); Liao et al. J Virol. Apr;87(8):4185-201 (2013).

[0093] Likewise, the design of gpl40 envelope forms is also well known in the art, along with the various specific changes which give rise to the gpl40C (uncleaved envelope), gp!40CF and gpl40CFI forms. Envelope gp!40 forms are designed by introducing a stop codon within the gp41 sequence. See Chakrabarti et al. at Figure 1 .

[0094] Envelope gpl40C refers to a gpl40 HIV-1 envelope design with a functional deletion of the cleavage (C) site, so that the gpl40 envelope is not cleaved at the furin cleavage site. The specification describes cleaved and uncleaved forms, and various furin cleavage site modifications that prevent envelope cleavage are known in the art. In some embodiments of the gp!40C form, two of the R residues in and near the furin cleavage site are changed to E, e.g., RRVVEREKR (SEQ ID NO: 9) is changed to ERVVEREKE (SEQ ID NO: 10), and is one example of an uncleaved gpl40 form. Another example is the gpl40C form which has the REKR site (SEQ ID NO: 8) changed to SEKS (SEQ ID NO: 11). See supra for references.

[0095] Envelope gpl40CF refers to a gpl40 HIV-1 envelope design with a deletion of the cleavage (C) site and fusion (F) region. Envelope gpl40CFI refers to a gpl40 HIV-1 envelope design with a deletion of the cleavage (C) site, fusion (F) and immunodominant (I) region in gp41 . See Chakrabarti et al. Journal of Virology vol. 76, pp. 5357-5368 (2002) at for example Figure 1, and Second paragraph in the Introduction on p. 5357; see Binley et al. supra for example at Abstract; see Gao et al. supra; see Liao et al. supra.

[0096] In certain embodiments, the envelope design in accordance with the present invention involves deletion of residues (e.g., 5-11, 5, 6, 7, 8, 9, 10, or 11 amino acids) at the N-terminus. For delta N-terminal design, amino acid residues ranging from 4 residues or even fewer to 14 residues or even more are deleted. These residues are between the maturation (signal peptide, usually ending with CXX, wherein X can be any amino acid) and "VPVXXXX. . . ", In case of CH505 T/F Env as an example, 8 amino acids (italicized and underlined in the below sequence) were deleted: MRVMGI0RNYP0WWIWSMLGFWMLMICNGA7WTFTFGVPVWKEAKTTLFCASDA KAYEKEVHNVWATHACVPTDPNPQE . .. (SEQ ID NO: 12) (rest of envelope sequence is indicated as

In other embodiments, the delta N-design described for CH505 T/F envelope can be used to make delta N-designs of other envelopes. In certain embodiments, the invention relates generally to an HIV-1 envelope immunogen, gpl60, gpl20, or gpl40, without an N-terminal Herpes Simplex gD tag substituted for amino acids of the N-terminus of gpl20, with an HIV leader sequence (or other leader sequence), and without the original about 4 to about 25, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. 21. 22. 23. 24. 25 amino acids of the N-terminus of the envelope (e.g. gpl20). See US2014/0248311, e.g. at paragraphs [0043] -[0050] , the contents of which publication is hereby incorporated by reference in its entire^.

[0097] The general strategy of deletion of N-terminal amino acids of envelopes results in proteins, for example gpl20s, expressed in mammalian cells that are primarily monomeric, as opposed to dimeric, and, therefore, solves the production and scalability problem of commercial gpl20 Env vaccine production. In other embodiments, the amino acid deletions at the N-terminus result in increased immunogenicity of the envelopes.

[0098] In certain aspects, the invention provides composition and methods which use a selection of Envs, as gpl20s, gpl40s cleaved and uncleaved, gpl45s. gpl50s and gpl60s, stabilized and/or multimerized trimers, as proteins, DNAs, RNAs, or any combination thereof, administered as primes and boosts to elicit an immune response. Envs as proteins could be co-administered with nucleic acid vectors containing Envs to amplify antibody induction. In certain embodiments, the compositions and methods include any immunogenic HIV-1 sequences to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic and/or consensus HIV-1 genes to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic group M and/or consensus genes to give the best coverage for T cell help and cytotoxic T cell induction. In some embodiments, the mosaic genes are any suitable gene from the HIV-1 genome. In some embodiments, the mosaic genes are Env genes, Gag genes, Pol genes, Nef genes, or any combination thereof. See e.g. US Patent No. 7951377. In some embodiments the mosaic genes are bivalent mosaics. In some embodiments the mosaic genes are trivalent. In some embodiments, the mosaic genes are administered in a suitable vector with each immunization with Env gene inserts in a suitable vector and/or as a protein. Tn some embodiments, the mosaic genes, for example as bivalent mosaic Gag group M consensus genes, are administered in a suitable vector, for example but not limited to HSV2, would be administered with each immunization with Env gene inserts in a suitable vector, for example but not limited to HSV-2.

[0099] In certain aspects the invention provides compositions and methods of Env genetic immunization either alone or with Env proteins to recreate the swarms of evolved viruses that have led to bnAb induction. Nucleotide-based vaccines offer a flexible vector format to immunize against virtually any protein antigen. Currently, two types of genetic vaccination are available for testing — DNAs and mRNAs.

[00100] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See Graham BS, Enama ME, Nason MC, Gordon IJ, Peel SA, et al. (2013) DNA Vaccine Delivered by aNeedle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for deliver}' of nucleic acids, as DNA and/or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments. DNA is formulated for deliver}' by a gene gun. In certain embodiments, DNA is administered by electroporation, or by a needle-free injection technologies, for example but not limited to Biojector® device. In certain embodiments, the DNA is inserted in vectors. The DNA is delivered using a suitable vector for expression in mammalian cells. In certain embodiments the nucleic acids encoding the envelopes are optimized for expression. Tn certain embodiments DNA is optimized, e.g. codon optimized, for expression. In certain embodiments the nucleic acids are optimized for expression in vectors and/or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus based vectors, rAdenovirus (e.g. Barouch DH, et al. Nature Med. 16: 319-23, 2010), recombinant mycobacteria (e.g. rBCG or M smegmatis) (Yu, JS et al. Clinical Vaccine Immunol. 14: 886- 093,2007; ibid 13: 1204-11,2006), and recombinant vaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010). for example but not limited to ALVAC, replicating (Kibler KV et al., PLoS One 6: e25674, 201 1 nov 9.) and non-replicating (Perreau M et al. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MV A)), adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons, Herpes Simplex Virus vectors, and other suitable vectors.

[00101] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies which contemplate using DNA or RNA, or may use complexes of nucleic acid molecules and other entities to be used in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer (amphiphilic block copolymer 704). See Cany et al.. Journal of Hepatology 2011 vol. 54 j 115-121; Amaoty et al., Chapter 17 in Yves Bigot (ed.). Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology⁷, vol. 859, pp293-305 (2012); Amaoty et al. (2013) Mol Genet Genomics. 2013 Aug;288(7-8):347-63. Nanocarrier technologies called Nanotaxi® for immunogenic macromolecules (DNA, RNA. Protein) delivery are under development. See for example nanocarrier technologies developed by In-Cell-Art. Nucleic acid can be delivered by injection and electroporation of muscle.

[00102] In certain aspects, the invention provides nucleic acids comprising sequences encoding envelopes of the invention. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[00103] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive antibodies. In certain embodiments, these are optionally formulated in lipid nanoparticles (LNPs). In certain embodiments, the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly-A tail, 5 ’cap.

[00104] In certain aspects the invention provides nucleic acids encoding the inventive envelopes. In non-limiting embodiments, the nucleic acids are mRNA. modified or unmodified, suitable for use any use, e.g. but not limited to use as pharmaceutical compositions. In certain embodiments, the nucleic acids are formulated in lipid, such as but not limited to LNPs.

[00105] In some embodiments the immunogens are administered as nucleic acids, including but not limited to mRNAs which could be modified and/or unmodified. See US Pub 20180028645A1, US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261 172, US Pub 20150038558, US Pub 20160032316, US Pub 20170043037, US Pub 20170327842, US Patent 10,006,007, US Patent 9,371,511, US Patent 9,012,219, US Pub 20180265848, US Pub 20170327842, US Pub 20180344838A1 at least at paragraphs [0260] -[0281], US Pub 20190153425 for non-limiting embodiments of chemical modifications, wherein each content is incorporated by reference in its entirety.

[00106] mRNAs delivered in LNP formulations have advantages over non-LNPs formulations. See US Pub 20180028645 Al, US Pub 20190274968, US Pub 20180303925, wherein each content is incorporated by reference in its entirety.

[00107] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[00108] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector.

[00109] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[00110] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the inventive sequences. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed from a DNA sequence encoding any one of the polypeptide sequence of the sequences of the invention, or a variant thereof or a fragment thereof. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of inventive envelopes. The RNA may be plus- stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.

[00111] In some embodiments, a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7- methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. In some embodiments, a RNA molecule useful with the invention may be single-stranded. In some embodiments, a RNA molecule useful with the invention may comprise synthetic RNA. [00112] The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity' of the envelope.

Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).

[00113] Methods for in vitro transfection of mRNA and detection of envelope expression are known in the art.

[00114] Methods for expression and immunogenicity determination of nucleic acid encoded envelopes are known in the art.

[00115] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. V arious methods for production and purification of recombinant proteins, including trimers such as but not limited to SOSIP based trimers, suitable for use in immunization are knoyvn in the art. In certain embodiments recombinant proteins are produced in CHO cells. [00116] It is readily understood that the envelope glycoproteins referenced in various examples and figures comprise a signal/leader sequence. It is well know n in the art that HIV- 1 envelope glycoprotein is a secretory protein with a signal or leader peptide sequence that is removed during processing and recombinant expression (without removal of the signal peptide, the protein is not secreted). See for example Li et al. Control of expression, glycosylation, and secretion of HIV-1 gpl20 by homologous and heterologous signal sequences. Virology⁷ 204(l):266-78 (1994) (“Li et al. 1994’'), at first paragraph, and Li et al. Effects of inefficient cleavage of the signal sequence of HIV- 1 gp!20 on its association with calnexin. folding, and intracellular transport. PNAS 93:9606-9611 (1996) (“Li et al. 1996”), at 9609. Any suitable signal sequence could be used. In some embodiments the leader sequence is the endogenous leader sequence. In other non-limiting examples, the leader sequence is human Tissue Plasminogen Activator (TP A) sequence, human CD5 leader sequence (e g. MPMGSLQPLATLYLLGMLVASVLA) (SEQ ID NO: 13). A skilled artisan appreciates that when used as immunogens, and for example when recombinantly produced, the amino acid sequences of recombinantly produced envelope immunogens do not comprise the signal/leader peptide sequences.

[00117] The immunogenic envelopes can also be administered as a protein prime and/or boost alone or in combination with a variety of nucleic acid envelope primes (e.g.. HIV -1 Envs delivered as DNA expressed in viral or bacterial vectors).

[00118] Dosing of proteins and nucleic acids can be readily determined by a skilled artisan. A single dose of nucleic acid can range from a few nanograms (ng) to a few micrograms (pg) or milligram of a single immunogenic nucleic acid. Recombinant protein dose can range from a few pg micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide.

[00119] Administration: The compositions can be formulated with appropriate carriers using known techniques to yield compositions suitable for various routes of administration. In certain embodiments the compositions are delivered via intramuscular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes, or any other suitable route of immunization.

[00120] The compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to 3M052, alum, poly IC, MF-59 or other squalene-based adjuvant, AS01B, or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen (Leroux- Roels et al., IABS Conference, April 2013). In certain embodiments, TLR agonists are used as adjuvants. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions.

[00121] In certain embodiments, the compositions and methods comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In non-limiting embodiments modulation includes PD- 1 blockade; T regulatory cell depletion; CD40L hyperstimulation; soluble antigen administration, wherein the soluble antigen is designed such that the soluble agent eliminates B cells targeting dominant epitopes, or a combination thereof. In certain embodiments, an immunomodulatory agent is administered in at time and in an amount sufficient for transient modulation of the subject's immune response so as to induce an immune response which comprises broad neutralizing antibodies against HIV- 1 envelope. Non-limiting examples of such agents is any one of the agents described herein: e.g. chloroquine (CQ), PTP1B Inhibitor - CAS 765317-72-4 - Calbiochem or MSI 1436 clodronate or any other bisphosphonate; a Foxol inhibitor, e g. 344355 Foxol Inhibitor, AS1842856 - Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, an agent which binds to a B cell receptor for a dominant HIV-1 envelope epitope, or any combination thereof. In non-limiting embodiments, the modulation includes administering an anti-CTLA4 antibody, OX-40 agonists, or a combination thereof. Non-limiting examples are of CTLA-1 antibody are ipilimumab and tremelimumab. In certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different.

[00122] Multimeric Envelopes

[00123] Presentation of antigens as particulates reduces the B cell receptor affinity necessary for signal transduction and expansion (see Batista et al. EMBO J. 2000 Feb 15; 19(4): 513-520). Displaying multiple copies of the antigen on a particle provides an avidity⁷ effect that can overcome the low affinity⁷ between the antigen and B cell receptor. The initial B cell receptor specific for pathogens can be low affinity, which precludes vaccines from being able to stimulate and expand B cells of interest. In particular, very few naive B cells from which HIV-1 broadly neutralizing antibodies arise can bind to soluble HIV-1 Envelope. Provided are envelopes, including but not limited to trimers as particulate, high-density array on liposomes or other particles, for example but not limited to nanoparticles. See, e.g. He et al. Nature Communications 7, Article number: 12041 (2016), doi : 10.1038/ncomms 12041; Bamrungsap et al. Nanomedicine, 2012, 7 (8), 1253-1271.

[00124] For development as a vaccine immunogen, multimeric nanoparticles that comprise and/or display HIV envelope protein or fragments on their surface can be used.

[00125] The nanoparticle immunogens are composed of various forms of HIV-envelope protein, e.g. without limitation envelope trimer. and self-assembling protein, e.g. without limitation ferritin protein. Any suitable ferritin could be used in the immunogens of the invention. In non-limiting embodiments, the ferritin is derived from Helicobacter pylori. In non-limiting embodiments, the ferritin is insect ferritin. In non-limiting embodiments, each nanoparticle displays 24 copies of the envelope protein on its surface.

[00126] Presenting multiple copies of antigens to B cells has been a longstanding approach to improving B cell receptor recognition and antigen uptake (see Batista et al. supra). The improved recognition of antigen is due to the avid interaction of multiple antigens with multiple B cell receptors on a single B cells, which results in clustering of B cells and stronger cell signaling. Furthermore, multimeric presentation improves antigen binding to mannose binding lectin which promotes antigen trafficking to B cell follicles. Selfassembling complexes comprising multiple copies of an antigen are one strategy⁷ of immunogen design approach for arraying multiple copies of an antigen for recognition by the B cell receptors on B cells (Kanekiyo. M.. Wei. C.J., Yassine, H.M., McTamney, P.M., Boyington, J.C., Whittle, J.R., Rao, S.S., Kong, W.P., Wang, L., and Nabel, G.J. (2013). Selfassembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499, 102-106; Ueda, G., Antanasijevic, A., Fallas, J.A., Sheffler, W., Copps, J., Ellis. D., Hutchinson, G.B., Moyer, A., Yasmeen, A., Tsybovsky, Y., et al. (2020). Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens. Elife).

[00127] In some instances, the gene of an antigen is fused via a linker/spacer to a gene of a protein which could self-assemble. Upon translation, a fusion protein is made that can selfassemble into a multimeric complex — also referred to as a nanoparticle displaying multiple copies of the antigen. In other instances, the protein antigen could be conjugated to the self- assembling protein via an enzymatic reaction, thereby forming a nanoparticle displaying multiple copies of the antigen. Non-limiting embodiments of enzymatic conjugation include without limitation sortase mediated conjugation. In some embodiments, linkers for use in any of the designs of the invention could be 2-50 amino acids long. e.g. 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids long. In certain embodiments, these linkers comprise glycine and serine amino acid in any suitable combination, and/or repeating units of combinations of glycine, serine and/or alanine. [00128] Ferritin is a well-known protein that self-assembles into a hollow particle composed of repeating subunits. In some species ferritin nanoparticles are composed of 24 copies of a single subunit, whereas in other species it is composed of 12 copies each of two subunits.

[00129] Non-limiting embodiments of sortase linkers could be used so long as their position allows multimerization of the envelopes. In a non-limiting embodiment, a C- terminal tag is LPXTG (SEQ ID NO: 14), where X signifies any amino acid but most commonly Ala, Ser, Glu, or a N-terminal pentaglycine repeat tag is added to the envelope trimer gene. In a non-limiting embodiment, a C-terminal tag is LPXTGG (SEQ ID NO: 15), where X signifies any amino acid but most commonly Ala, Ser. Glu.

[00130] To improve the interaction between the naive B cell receptor and immunogens, in some embodiments, the envelope design is created so the envelope is presented on particles, e.g. but not limited to nanoparticle. In some embodiments, the HIV-1 Envelope trimer could be fused to ferritin. Ferritin protein self assembles into a small nanoparticle with three-fold axis of symmetry. At these axes the envelope protein is fused. Therefore, the assembly of the three-fold axis also clusters three HIV-1 envelope protomers together to form an envelope trimer. Each ferritin particle has 8 axes which equates to 8 trimers being displayed per particle. See e.g. Sliepen et al. Retrovirology 2015 12:82, DOI: 10.1186/sl2977-015-0210-4. [00131] Any suitable ferritin sequence could be used. In non-limiting embodiments, ferritin sequences are disclosed in US2019/0330279, the content of w hich is hereby incorporated by reference in its entirety.

[00132] Ferritin nanoparticle linkers: The ability to form HIV-1 envelope ferritin nanoparticles relies self-assembly of 24 ferritin subunits into a single ferritin nanoparticle. The addition of a ferritin subunit to the C-terminus of HIV- 1 envelope may interfere with the ability of the ferritin subunit to fold properly and or associate with other ferritin subunits. When expressed alone ferritin readily forms 24-subunit nanoparticles, however appending it to envelope only yields nanoparticles for certain envelopes. Since the ferritin nanoparticle forms in the absence of envelope, the envelope could be sterically hindering the association of ferritin subunits. Thus, ferritin can be designed with elongated glycine-serine linkers to further distance the envelope from the ferritin subunit. To make sure that the glycine linker is attached to ferritin at the correct position, constructs can be created that attach at second amino acid position or the fifth amino acid position. The first four n-terminal amino acids of natural Helicobacter pylori ferritin are not needed for nanoparticle formation but may be critical for proper folding and oligomerization when appended to envelope. Thus, constructs can be designed with and without the leucine, serine, and lysine amino acids following the glycine-serine linker. The goal will be to find a linker length that is suitable for formation of envelope nanoparticles when ferritin is appended to most envelopes. Any suitable linker between the envelope and ferritin could be uses, so long as the fusion protein is expressed and the trimer is formed.

[00133] Another approach to multimerize expression constructs uses Staphylococcus sortase A transpeptidase ligation to conjugate inventive envelope trimers, for example but not limited to cholesterol. The trimers can then be embedded into liposomes via the conjugated cholesterol. To conjugate the trimer to cholesterol either a C-terminal LPXTG (SEQ ID NO: 14) tag or a N-terminal pentaglycine repeat tag is added to the envelope trimer gene. Cholesterol is also synthesized with these two tags. Sortase A is then used to covalently bond the tagged envelope to the cholesterol. The sortase A-tagged trimer protein can also be used to conjugate the trimer to other peptides, proteins, or fluorescent labels. In non-limiting embodiments, the sortase A tagged trimers are conjugated to ferritin to form nanoparticles. [00134] The invention provides design of envelopes and trimer designs wherein the envelope comprises a linker which permits addition of a lipid, such as but not limited to cholesterol, via a sortase A reaction. See e.g. Tsukiji. S. and Nagamune, T. (2009), Sortase- Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering. ChemBioChem, 10: 787-798. doi:10.1002/cbic.200800724; Proft, T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett (2010) 32: 1. doi: 10.1007/sl0529-009-0116-0; Lena Schmohl, Dirk Schwarzer. Sortase-mediated ligations for the site-specific modification of proteins, Cunent Opinion in Chemical Biology, Volume 22, October 2014, Pages 122-128, ISSN 1367-5931, dx.doi.org/10.1016/j.cbpa.2014.09.020; Tabata et al. Anticancer Res. 2015 Aug;35(8):441 1 - 7; Pritz et al. J. Org. Chem. 2007, 72, 3909-3912.

[00135] The lipid modified envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[00136] The lipid modified and multimerized envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[00137] Non-limiting embodiments of envelope designs for use in sortase A reaction are shown in Figure 24 B-D of US2020/0002383, incorporated by reference in its entirety.

[00138] Table 1 shows a summary of envelope sequences disclosed herein. See Example 1 and Figures 1 and 2.

[00139] Table 2 shows a summary of additional modifications that can be incorporated into the JR-FL MCD5 envelope described herein. Alternatively, Table 2 discloses exemplary sequences from the CH848 infected individual and variants thereof that can comprise the modification to the VI loop described herein. In some embodiments, the recombinant HIV-1 envelope comprises any envelope sequence from the CH848 infected individual and variants thereof (e.g., in Table 2) comprising HEKGG (SEQ ID NO: 7) at positions 134-138 (HXB2 numbering).

[00140] DH270 light chain binds to N301 glycan. In some embodiments, a N301 gly site is used (e.g. change #2 in row 5 of Table 2, supra).

[00141] DH270 heavy chain binds to N332 glycan. In some embodiments, a N332 gly site is used (e.g. changes #4 and #5 in row 5 of Table 2, supra).

[00142] V3 glycan Abs bind GDIR (SEQ ID NO: 5). In some embodiments, a change #3 to “GDIR” (SEQ ID NO: 5) is needed (e.g. “GDIR” (SEQ ID NO: 5) sequence in row 5 of Table 2, supra).

[00143] GDIR/Kmotif: V3-glycan broadly neutralizing antibodies typically contact the c- terminal end of the third variable region on HIV-1 envelope. There are four amino acids, Gly324, Asp325, Ile326, and Arg327, bound by V3-glycan neutralizing antibodies. While Arg327 is highly conserved among HIV-1 isolates, Lys327 also occurs at this site. The CH848.3.D0949.I0.17 isolate naturally encodes the less common Lys327. In contrast to CH848.3.D0949.10.17 with the Lys327, the precursor antibody of the DH270 V3-glycan broadly neutralizing antibody lineage barely binds to CH848.3.D0949.10.17 encoding Arg327. Thus, Arg327 is useful for the precursor to bind and the lineage of neutralizing antibodies to begin maturation. However, somatically mutating antibodies on the path to developing neutralization breadth bind better to Env encoding Arg327. Thus, Env must encode Lys327 to initiate DH270 lineage development. However, to best interact with affinity maturing DH270 lineage members the Env should encode Arg327. Thus, a plausible vaccine regimen to initiate and select for developing bnAbs would include a priming immunogen encoding, Lys327 and a boosting immunogen encoding Arg327. The Arg327 boosting immunogen w ould optimally target the affinity maturing DH270 lineage members, while not optimally binding the DH270 antibodies that lack affinity maturation. Non-limiting embodiments of vaccination regimens could include: priming wdth CH848.3.D0949.10.17 based envelope design also with Lys327, followed by administering of CH848.3.D0949.10.17 based envelope design with Arg327. Non-limiting embodiments of vaccination regimens could include: priming with 19CV3 based envelope design also with Lys327, followed by administering of CH848.3.D0949.10. 17 based envelope design with Arg327.

[00144] The name CH848.d0949.10.17 DT can be used interchangeably with CH848.d0949.10.17.N133D.N138T. The name CH848.d0949.10.17 can be used interchangeably with CH848.d0949. 10. 17WT. In certain embodiments, CH848.d0949.10.17DT envelope comprises additional modifications D230N.H289N.P291S.E169K and is referred to as CH848.d0949.10. 17 DTe. In certain embodiments, CH848.d0949.10.17 envelope comprises additional modifications D230N.H289N.P291S.E169K and is referred to as CH848.d0949.10.17WTe.

[00145] The invention contemplates any other design, e.g. stabilized trimer, of the sequences described here in. For non-limiting embodiments of additional stabilized trimers see US2015/0366961, US2020/0002383, US2021/0187091 and US2020/0113997, and F14 and/or VT8 designs (US2021/0379177) all of which are incorporated by reference in their entirety.

[00146] Any suitable signal peptide could be used. In designs comprising ferritin for multimerization, any suitable linker could be used betw een the envelope sequence and a ferritin sequence. EXAMPLES

[00147] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[00148] Example 1

[00149] Immunogen design to target the functional improbable mutation S27Y in the DH270 lineage

[00150] DH270 UCA heterozygous heavy chain variable and light chain variable region double knock-in mice CVHDHJH . V/.J,. KI strain) were generated as previously described [Science 2019], Briefly, DH270 UCA KI mice administered protein immunogens were immunized intramuscularly and/or subcutaneously with 25 mcg of protein immunogen along with 5 mcg of the TLR4 agonist-based adjuvant GLA-SE (IDRI). All mice were cared for in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All study protocol and all veterinarian procedures were approved by the Duke University Institutional Animal Care and Use Committee (IACUC).

[00151] Antigen-specific single B cell sorting, antibody isolation and antibody sequencing was performed as previously described [Science 2019], Memory B cells from splenocytes were sorted by fluorescence activated cell sorting using a panel of fluorochrome-antibody conjugates (all from BD Biosciences): FITC anti-mouse IgGl (A85-1), FITC anti-mouse IgG2a/2b (R2-40), FITC anti-mouse IgG3 (R40-82), PE anti-mouse GL7 (GL7), PE-Cy7 anti-mouse IgM (R6-60.2), AlexaFluor700 anti-mouse CD19 (1D3), BV510 anti-mouse IgD (1 l-26C.2a). and BV650 anti-mouse B220 (RA3-6B2). Cells were also labeled with biotinylated SOSIP envelope trimers (10.17 or 10.17DT) conjugated to streptavidin-BV421 or streptavidin- AF647. Env-specific memory' B cells were identified as viable B220+CD19+IgM-IgD-GL7-IgGl/2/3+ cells that bound both BV421- and AF647-conjugated SOSIP trimers. Single cells were sorted on a BD FACS Ariall into 96-well PCR plates containing lysis buffer. Plates were immediately snap frozen and stored at -80° C. [00152] Immunoglobulin genes were amplified as previously described with some modifications. Immunoglobulin genes from a single B cell were reverse transcribed with Superscript III (ThermoFisher) using random hexamer oligonucleotides as primers. The complementary’ DNA was used to perform nested PCR for DH270 heavy and light chain genes using AmpliTaq gold (ThermoFisher) and primers designed to bind to the DH270 UCA variable region sequence. In parallel, PCR reactions were done with mouse immunoglobulinspecific primers. PCR amplicons were identified by gel electrophoresis and purified for Sanger sequencing using a PCR clean-up kit (Qiagen). Contigs of the PCR amplicon forward and reverse sequences were made, and immunogenetics annotation was performed with Cloanalyst using both the human and mouse Ig gene libraries.

[00153] The ability’ of CH848.D0949.10.17DT and CH848.D0949.10.17 immunogens was evaluated to select for these four mutations in DH270 unmutated common ancestor (UCA) antibody knock-in mice. Immunizations of these mice generated antibodies with 3 of the four desired mutations. However, none of the antibodies had all four mutations. The antibody mutation that was lacking in most cases was the mutation that encodes the S27Y amino acid change (Figure 1).

[00154] Antibody -mediated HIV-1 neutralization was measured using Tat-regulated luciferase (Luc) reporter gene expression to quantify reductions in virus replication in TZM- bl cells as described previously. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. The monoclonal antibody was pre-incubated with virus (-150,000 relative light unit equivalents) for 1 h at 37 °C, and TZM-bl cells were subsequently added. After 48 h cells were lysed and Luc activity determined using a microtiter plate luminometer and BriteLite Plus Reagent (Perkin Elmer). Neutralization titers are the inhibitory' concentration at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells (IC50 respectively).

[00155] The lack of S27Y is significant as this substitution in the antibody variable region can lead to an increase in the neutralization breadth and potency of DH270 antibodies. Thus, S27Y plays an important role in monoclonal antibody neutralization breadth in combination with other mutations (Figure 2).

[00156] When the S27Y was added to a vaccine-induced antibody called MU89, the neutralization potency and breadth of the antibody increased beyond that seen by’ early DH270 lineage antibodies (Figure 2). Thus, an immunogen that is capable of selecting for the S27Y substitution in DH270 antibodies was designed. Mammalian cell display of envelope variants was used to select for an envelope that binds with high affinity to the DH270 UCA antibody with S27Y but binds lowly to DH270 precursor antibody without the S27Y substitution.

[00157] To this end, HIV-1 envelope from the JR-FL isolate, which was not bound by the DH270 UCA (Figure 3) was used. A site-saturation library⁷ was designed using JRFL as a template (PMID: 31806786). The site-saturation envelope library was synthesized (GenScript) to contain envelopes with a single substitution. The substitutions introduced could be any of the other 19 possible amino acids at any one position within the gpl20 subunit. The stabilized, cleaved HIV-1 envelope gpl40 was fused to a C-terminal c-myc tag and HRV-3C cleavage site, followed by a platelet-derived growth factor receptor transmembrane region that ensured cell surface presentation of the envelope. The open reading frame encoding the envelope library⁷ was cloned into the mammalian expression vector VRC8400 CMV/R.

[00158] Sixteen micrograms of CMV/R vector carrying HIV-1 SOSIP library were cotransfected with 67 pg of carrier DNA (CMV/R vector) without the insert and 3.7 pg of plasmid coding Furin into Freestyle 293F cells using a 293fectin Transfection Kit (Thermo Scientific) according to manufacturer’s instruction. Cells were cultured for 48 hours with shaking at 120 rpm in an incubator filled with 8% CO2 at 37°C. Tw enty⁷ million harvested transfected cells were stained with 500 pl of 100 pg/ml DH270 UCA with the S27Y substitution and 10 pg/ml chicken anti-c-myc. Sorted cells were sequenced by tagmentation and illumina miseq sequencing.

[00159] Three rounds of sorting cells that bound to DH270 UCA+S27Y (Figure 4) was performed. There was a clear binding population of cells after three rounds of sorting (Figure 4). These sorted cells were tested for binding to DH270 UCA3+S27Y. Confirming the sorting experiments, the cell population expressing envelope in culture bound to DH270 UCA S27Y, whereas the starting wildty pe JR-FL Env did not bind (Figure 5). Transfected cells w ere stained with 500 pl of up to 100 pg/ml DH270 UCA with the S27Y substitution and 10 pg/ml chicken anti-c-myc. Stained cells were analysed on a LSRII flow cytometer.

[00160] The binding was specific as DH270 UCA+S27Y bound in a dose-dependent manner to the selected cells, and DH270 UC A+S27Y did not bind to cells not expressing any Env (Figure 5). Next, an examination was conducted to determine whether any of the other 3 critical mutations (R98T, G57R, and L48Y) could confer binding to the selected population of cells expressing the JR-FL Env variant (Figure 6). Transfected cells were stained with 500 pl of upto 100 pg/ml DH270 UCA with the S27Y substitution and 10 pg/ml chicken anti-c- myc. Stained cells were analyzed on a LSRII flow cytometer.

[00161] DH270 UCA with a G57R substitution added bound to both the wildtype JR-FL

Env displayed on the surface of cells, but bound better to the Env variant expressed by sorted cells. DH270 UCA+ R98T exhibited dose-dependent binding to the selected Env variant as well (Figure 6). Lastly, binding to DH270 UCA+L48Y was weak for both the wildtype and selected JR-FL Env.

[00162] To identify the amino changes that conferred the improved binding to DH270 antibodies with the critical improbable mutations, MiSeq next generation sequencing of the env genes expressed in selected cell population (Figure 7) was performed. Sorted cells were sequenced by tagmentation and illumina miseq sequencing. Sequences were analyzed by an in house bioinformatics pipeline and visualized in Geneious software.

[00163] The NGS results showed that VNATN (SEQ ID NO: 16) at positions 134 - 138 had mutated to HEKGG (SEQ ID NO: 7) in greater than 99% of the sequence reads from the sorted cells (Figure 7). Soluble recombinant HIV-1 JR-FL Env trimers with these substitutions w ere prepared. JR-FL_MCD5 is a short term for mammalian cell display 5 amino acid changes. Using the soluble JR-FL_MCD5 envelope, the binding magnitude for DH270 antibodies to JR-FL MCD5 by ELISA and biolayer interferometry (BLI) was determined (Figures 8 and 9). Monoclonal antibody ELISA binding assays were performed as described previously. In brief, Nuncsorp plates were coated with HIV-1 envelope, washed and blocked. After blocking was complete, a dilution series of antibodies starting at 100 pg mL'¹ was incubated in triplicate wells for 90 min. monoclonal antibodies were incubated with the Env in triplicate as positive controls for blocking. After 90 min the antibody was washed away. Binding by monoclonal antibodies was determined with a 1:8000 dilution of anti-IgG Fc (HRP)-conjugate. HRP was detected with tetramethylbenzidine and stopped with 1% HC1. The absorbance at 450 nm of each w ell was read with a Spectramax plate reader (Molecular Devices).

[00164] In ELISA, the DH270 UCA bound weakly to the JR-FL MCD5, but not to the wildtype Env. DH270 UCA+S27Y only bound strongly to the JR-FL MCD5. When all four mutations were present in the DH270 antibody there was no difference in binding between wildtype and MCD5 suggesting the MCD5 envelope provides a selective advantage to early DH270 antibodies with only of the critical mutations (Figure 8). The JR-FL MCD5 also bound stronger to DH270 UCA+G57R but the difference was modest. Similar to the binding on the cell surface, DH270 UCA+L48Y bound markedly better to JR-FL MCD5 than wildtype JR-FL (Figure 9). Biolayer interferometry was performed as previously described. BLI ligand titration and binding kinetics experiments were conducted on the Octet Red96e system (Sartorius) at 30°C and an orbital shake speed of lOOOrpm. All assays used 0.22pm filtered PBS buffer supplemented with 0.05% Tween 20 and 0.1% bovine serum albumin (PBS-T-BSA) and flat bottom 96-well plates (Greiner). For the ligand titration experiment, two-fold serial dilutions of the biotinylated Env trimers, with a starting concentration of lOpg/mL, were immobilized on hydrated Streptavidin (SA) Tips (Octet® Sartorius) for 120 seconds. After a 60 second wash and 180 second Baseline step in PBS-T-BSA, the tips were incubated with 2000nM of DH270UCA Fab, 500nM of Mu89 Fab and 500nM of Mu89+S27Y Fab for 600 seconds. The optimal Env concentrations for affinity studies were analyzed from the sensorgram traces and binding responses using Data Analysis HT 12.0 softw are (Forte Bio). For the binding kinetics experiment, the chosen Env concentrations were immobilized on hydrated SA biosensor tips and incubated with t wo- fold serial dilutions of antibody Fabs for 600 seconds followed by a 600-second-long dissociation step in PBS-T- BSA. The binding response sensorgram curves were globally fitted using a 1 : 1 Binding Model and the rate constants k_a, ka and Ka were calculated using Data Analysis HT 12.0 software (Forte Bio).

[00165] Overall, the binding show ed that JR-FL MCD5 bound better to DH270 minimally mutated antibodies than wildty pe JR-FL with the largest difference for antibodies with S27Y or R98T. The binding kinetics showed a similar trend with JR-FL MCD5 binding better to DH270 antibodies than wildtype JR-FL (Figure 9). More specifically, DH270 UCA+S27Y bound with 38 nM apparent affinity and 549 nM affinity to the JR-FL MCD5 (Figure 9). [00166] Without being bound by theory, it is hypothesized that the optimized binding to DH270 antibodies would enable JR-FL MCD5 to bind to DH270 antibodies in serum more avidly than JR-FL. To determine whether the natural DH270 lineage antibodies could react with DH270, CH848, the individual who made the DH270 antibody lineage, serum IgG binding throughout the course of infection w as assessed (Figure 10). Biotinylated SOSIP capture ELISAs were performed as previously described. Two pg mL'¹ of streptavidin in sodium bicarbonate buffer was incubated in sealed Nunc-absorp (ThermoFisher) plates overnight at 4 °C. Unbound protein was washed away and the plates were blocked with SuperBlock for 1 h. Envelope was incubated in the plate for Ih and unbound protein was washed away. Serial dilution of serum or monoclonal antibodies were added to the plate for 90 min. Binding antibodies were detected with FIRP-labeled anti-IgG Fc antibody. HRP was detected with 3,3',5,5'-Tetramethylbenzidine. Binding titers were analyzed as area-under- curve of the log-transformed concentrations.

[00167] JR-FL MCD5 Env trimer bound weaker to serum than wildtype Env until August 2011. After this timepoint JR-FL bound better to serum IgG than the wildtype JR-F1 Env (Figure 10). Binding antibodies were detected with HRP-labeled anti-IgG Fc antibody. HRP was detected with 3,3',5,5'-Tetramethylbenzidine. Binding titers were analyzed as area-under- curve of the log-transformed concentrations.

[00168] Antibody -mediated HIV- 1 neutralization was measured using Tat-regulated luciferase (Luc) reporter gene expression to quantify reductions in virus replication in TZM- bl cells as described previously. This timepoint corresponded with time at which serum neutralization breadth began to increase (Figure 11). We performed next generation sequencing of the B cell repertoire at these timepoints and saw that DH270 sequences were present at February and June 2012 (Figure 12). Sequencing was done as described in Bonsignori et al. Science translational medicine 2017.

[00169] Thus, increases in JR-FL MCD5 binding was associated with the emergence of the DH270 lineage.

[00170] In some embodiments, to be an optimal boosting immunogen in a series of envelope immunizations, the JR-FL MCD5 should bind strongly to mouse serum IgG generated by the preceding Envs in the sequential series of Env immunogens. To determine whether MCD5 would be an optimal boost immunogen for antibodies that have generated with the prime and boost immunogens, ELISAs with serum from vaccinated DH270 UCA knock in mice w ere performed. The mouse serum bound to JR-FL MCD5 indicating there were B cells expressing antibodies that would be engaged by JR-FL MCD5 (Figure 13). Ongoing studies are testing JR-FL MCD5 as the second or third Env in the sequential vaccine (Figure 14). [00171] Example 2

[00172] This example describes animal studies with HIV-1 envelopes designed to prime and boost V3 glycan antibodies lineages.

[00173] The envelopes described in Table 1, expressed as recombinant proteins or modified mRNA formulated in LNP, are analyzed in animal studies including mouse and NHP animal models. The mouse animal model could be any model, including an animal model comprising a DH270UCA transgene.

[00174] An object of the present invention was to generate an envelope that can select for B cells with S27Y substitutions in DH270 antibodies. As shown in the accompanying figures herein, in vitro engineered Env JRFL.MCD5 displayed an increase in binding titer and affinity to DH270UCA S27Y compared to wildtype JRFL Env.

[00175] In vivo experiments with DH270 UCA knock-in mouse immunizations (Figure 15) with MCD5 elicited autologous and heterologous neutralization breadth in the serum (Figures 16 and 17). Antibody-mediated HIV-1 neutralization was measured using Tat- regulated luciferase (Luc) reporter gene expression to quantify reductions in virus replication in TZM-bl cells as described previously. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. The serum was pre-incubated with virus (-150.000 relative light unit equivalents) for 1 h at 37 °C, and TZM-bl cells were subsequently added. After 48 h cells were lysed and Luc activity determined using a microtiter plate luminometer and BriteLite Plus Reagent (Perkin Elmer). Neutralization titers are the inhibitory dilution at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells (ID50).

[00176] MCD5 trimer boosting immunizations increased selection of S27Y alone (Figure 18) or in combination with other key light chain mutations compared to a CH848 envelope regimen (Figure 18). Bulk single chain BCR repertoire sequencing of mouse splenocytes was performed as previously described [Mu et al 2022], Briefly, next-generation sequencing (NGS) was performed on mouse antibody heavy and light chain variable genes using the Illumina MiSeq platform. RNA was purified from splenocytes using a RNeasy Mini Kit (Qiagen, Cat# 74104). Purified RNA was quantified via Nanodrop (Thermo Fisher Scientific) and used to generate Illumina-ready heavy and light chain sequencing libraries using the SMARTer Mouse BCR IgG H/K/L Profiling Kit (Takara, Cat# 634422). Briefly, 1 pg of total purified RNA from splenocytes was used for reverse transcription with Poly dT provided in the SMARTer Mouse BCR kit for cDNA synthesis. Heavy and light chain genes were then separately amplified using a 5‘ RACE approach with reverse primers that anneal in the mouse IgG constant region for heavy chain genes and IgK for the light chain genes (SMARTer Mouse BCR IgG H/K/L Profiling Kit). While the DH270 UCA uses a lambda light chain (VL2-23), the UCA KI mouse model has the light chain gene knocked into the kappa locus, therefore kappa primers provided in the SMARTer Mouse BCR kit were used for light chain gene library preparation for both the DH270 UCA KI and CH235 UCA KI mice. 5 pl of cDNA was used for heavy and light chain gene amplification via two rounds of PCR; PCRI used 18 cycles and PCR2 used 12 cycles. During PCR2, Illumina adapters and indexes were added. Illumina-ready sequencing libraries were then purified and size-selected by AMPure XP (Beckman Coulter, Cat# A63881) using kit recommendations. The heavy and light chain libraries per mouse were indexed separately, thus allowing us to deconvolute the mousespecific sequences during analysis. Libraries were quantified using QuBit Fluorometer (Thermo Fisher). Mice were pooled by groups for sequencing on the Illumina MiSeq Reagent Kit v3 (600 cycle) (Illumina, Cat# MS-102-3003) using read lengths of 301/301 with 20% PhiX.

[00177] DH270 antibodies isolated from MCD5 boosted mice demonstrated the first instance of vaccine induction of DH270 antibodies that have all 4 key mutations (Figure 27). Immunogens specifically engineered or selected to have high affinity' for a particular antibody can select for critical mutations, thereby enabling mutation-guided vaccine design. Vaccine regimens may be able to be shortened using these engineered envelopes and optimal deliveryplatforms like nanoparticles or mRNA-LNPs.

[00178] The envelopes in Table 1 will be produced under cGMP conditions as a recombinant protein and/or mRNA formulated in LNP for use in Phase I clinical trial.

SEQUENCES

>HV1303395

MPMGSLQPI.ATT,YIJ,GMI,VASVT,AVFKLWVTVYYGVPVWKEACTTT,FCASDAKAYDTKVR

NVWATHCCVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLC

VTLNCKDHEKGGTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNN

T S YRLISCDT S VITQACPKI SFEPIPIH YC APAGFAILKCNDKTFNGKGPCKNV ST VQCT

HGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIH

IGPGRWFYTTGEIIGDIRQAHCNISRAKWNDTLKQIVIKLREQFENKTIVFNHSSGGDPE

IVMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNTITLPCRIKQIINMWQEIGKAM

YAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPL

GVAPTKCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQ

NNCLRAPECQQRMLQLTVWGIKQLQARVLAVERYLGDQQLLGIWGCSGKLICCTAVPWNA

SWSNKSLDRIWNNMTWMEWEREIDNYTSEIYTLIEESQNQQEKNEQELLELDGGGLVPQQ

SGGLNDIFEAQKIEWHEG** (SEQ ID NO: 1)

Signal peptide: MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO: 13)

Avi tag and linker: GGGLVPQQSGGLNDIFEAQKIEWHEG (SEQ ID NO: 17)

>HV1303396

MPMGSLQPLATLYLLGMLVASVLAVEKLWVTVYYGVPVWKEACTTLFCASDAKAYDTKVR

NVWATHCCVPTDPNPQEVVLENVTEHFNMWKNNMVEQMQEDIISLWDQSLKPCVKLTPLC

VTLNCKDHEKGGTTNDSEGTMERGEIKNCSFNITTSIRDKVQKEYALFYKLDVVPIDNNN

TSYRLISCDTSVITQACPKISFEPIPIHYCAPAGFAILKCNDKTFNGKGPCKNVSTVQCT

HGIRPVVSTQLLLNGSLAEEEVVIRSDNFTNNAKTIIVQLKESVEINCTRPNNNTRKSIH

IGPGRWF YTTGEIIGDIRQ AHCNI SRAKWNDTLKQI VIKLREQFENKTI VFNH SSGGDPE

1VMHSFNCGGEFFYCNSTQLFNSTWNNNTEGSNNTEGNT1TLPCR1KQ11NMWQE1GKAM

YAPPIRGQIRCSSNITGLLLTRDGGINENGTEIFRPGGGDMRDNWRSELYKYKVVKIEPL

GVAPTKCKRRVVGRRRRRRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQ

NNCLRAPECQQRMLQLTVWGIKQLQARVLAVERYLGDQQLLGIWGCSGKLICCTAVPWNA

SWSNKSLDRIWNNMTWMEWEREIDNYTSEIYTLIEESQNQQEKNEQELLELDLPETGG**

(SEQ ID NO: 2)

Signal peptide: MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO: 13) Sortase A tag: LPETGCT (SEQ ID NO: 18)

>HV1303395

G r \:C< GGTGGAAAAGCTGTGGGTCACCGTGTACTACGGCGTGCCTGTGTGGAAGGAAGCTTGTAC CACACTCTTCTGTGCCAGCGACGCCAAGGCCTACGACACCAAGGTGCGGAACGTGTGGGCCACCC

ACTGTTGCGTGCCGACCGATCCTAACCCCCAGGAGGTGGTGCTGGAAAATGTCACCGAGCACTTCA ACATGTGGAAGAACAATATGGTGGAGCAGATGCAGGAGGACATCATCTCCCTGTGGGACCAGAGC CTGAAGCCTTGTGTGAAACTGACACCTCTGTGCGTGACCCTGAACTGCAAGGACCACGAGAAGGG CGGAACAACAAACGACTCTGAGGGCACAATGGAAAGAGGCGAGATCAAAAATTGCAGCTTTAAC

ATCACAACAAGTATCAGAGATAAGGTCCAGAAAGAATACGCCCTGTTCTACAAGCTGGATGTGGT GCCTATCGACAACAACAATACCAGCTACAGACTGATCAGCTGCGACACCTCTGTGATCACCCAGG CCTGCCCCAAGATCAGCTTTGAGCCTATCCCCATCCACTACTGCGCCCCAGCCGGCTTCGCCATTCT

GAAGTGCAATGATAAGACCTTCAACGGCAAGGGCCCCTGCAAAAACGTGAGCACCGTGCAGTGCA CCCACGGCA’l TCGCCCTGTGGTGAGCACACAGCTGCTGCTGAACGGATCrCTGGCCGAGGAGGAA

GTGGTGATTAGAAGCGATAACTTCACCAACAACGCCAAAACAATCATCGTGCAGCTGAAGGAAAG

CGTGGAAATCAACTGTACCCGGCCTAATAACAACACAAGAAAGTCTATCCACATCGGTCCTGGCC

GGTGGTTCTACACCACCGGAGAAATCATCGGCGACATCAGACAGGCTCATTGCAACATCAGCAGA GCTAAGTGGAACGATACCCTGAAGCAGATCGTGATTAAGCTCAGAGAGCAATTTGAGAACAAGAC

AATCGTGTTCAACCACTCTTCTGGAGGCGACCCCGAGATCGTGATGCACTCCTTCAACTGCGGCGG CGAATTCTTCTATTGTAACAGCACGCAGCTGTTCAACAGCACCTGGAACAATAACACCGAGGGCA GCAACAACACCGAGGGCAACACCATCACCCTGCCTTGCAGAATCAAGCAGATCATCAATATGTGG

CAGGAGATCGGCAAAGCCATGTATGCCCCTCCAATCCGGGGCCAGATCAGATGCAGCAGCAACAT CACAGGACrGCTGCTGACAAGAGArGGCGGAArCAATGAGAACGGCACCGAAATCTl TAGACCCG

GCGGCGGCGACATGCGGGACAACTGGCGCAGCGAGCTGTACAAGTACAAAGTGGTGAAGATCGA

GCCCCTGGGAGTGGCCCCAACAAAGTGTAAAAGAAGAGTGGTGGGCAGACGGAGACGGAGACGG GCCGrCGGCATCGGCGCTGri TrCCTGGGCTTCCTGGGCGCCGCrGGCAGCACCATGGGCGCCGCC TCTATGACCCTGACCGTTCAGGCTAGACAGCTGCTGAGCGGCATCGTGCAGCAGCAAAACAATTG CCTGAGAGCCCCTGAGTGCCAGCAAAGAATGCTGCAGCTGACCGTCTGGGGCATTAAGCAACTGC AAGCCAGGGTGCrCGCrGTGGAGAGATACCTGGGCGATCAGCAGCTGCTGGGCATCTGGGGArGT

AGCGGCAAGCTGATCTGCTGCACCGCCGTGCCTTGGAACGCCAGCTGGTCCAACAAGAGCCTGGA CAGAATCTGGAACAATATGACATGGATGGAATGGGAGCGGGAAATCGACAACTACACCAGTGAA

ATCrACACACTGATCGAAGAAAGCCAAAACCAGCAGGAGAAGAA'IGAGCAGGAGCTGCTGGAGC TGG ACGG G < A GGG Cl Gi G G < GAG A GA A< AG< A> X; < G ■ GAG GGAAGG AG AAG GG GA/AG

A : CAd GiG A€G. CAGGC lAAl GA (SEQ ID NO: 3)

>HV1303396

GJ G GCC GTGGAAAAGCTGTGGGTCACCGTGTACTACGGCGTGCCTGTGTGGAAGGAAGCTTGTAC

CACACTCTTCTGTGCCAGCGACGCCAAGGCCTACGACACCAAGGTGCGGAACGTGTGGGCCACCC

ACTGTTGCGTGCCGACCGATCCTAACCCCCAGGAGGTGGTGCTGGAAAATGTCACCGAGCACTTCA

ACATGTGGAAGAACAATATGGTGGAGCAGATGCAGGAGGACATCATCTCCCTGTGGGACCAGAGC

CTGAAGCCTTGTGTGAAACTGACACCTCTGTGCGTGACCCTGAACTGCAAGGACCACGAGAAGGG

CGGAACAACAAACGACTCTGAGGGCACAATGGAAAGAGGCGAGATCAAAAATTGCAGCTTTAAC

ATCACAACAAGTATCAGAGATAAGGTCCAGAAAGAATACGCCCTGTTCTACAAGCTGGATGTGGT

GCCTATCGACAACAACAATACCAGCTACAGACTGATCAGCTGCGACACCTCTGTGATCACCCAGG

CCTGCCCCAAGATCAGCTTTGAGCCTATCCCCATCCACTACTGCGCCCCAGCCGGCTTCGCCATTCT

GAAGTGCAATGATAAGACCTTCAACGGCAAGGGCCCCTGCAAAAACGTGAGCACCGTGCAGTGCA

CCCACGGCATTCGCCCTGTGGTGAGCACACAGCTGCTGCTGAACGGATCTCTGGCCGAGGAGGAA

GTGGTGATTAGAAGCGATAACTTCACCAACAACGCCAAAACAATCATCGTGCAGCTGAAGGAAAG

CGTGGAAATCAACTGTACCCGGCCTAATAACAACACAAGAAAGTCTATCCACATCGGTCCTGGCC

GGTGGTTCTACACCACCGGAGAAATCATCGGCGACATCAGACAGGCTCATTGCAACATCAGCAGA

GCTAAGTGGAACGATACCCTGAAGCAGATCGTGATTAAGCTCAGAGAGCAATTTGAGAACAAGAC AATCGTGTTCAACCACTCTTCTGGAGGCGACCCCGAGATCGTGATGCACTCCTTCAACTGCGGCGG

CGAATTCTTCTATTGTAACAGCACGCAGCTGTTCAACAGCACCTGGAACAATAACACCGAGGGCA

GCAACAACACCGAGGGCAACACCATCACCCTGCCTTGCAGAATCAAGCAGATCATCAATATGTGG

CAGGAGATCGGCAAAGCCATGTATGCCCCTCCAATCCGGGGCCAGATCAGATGCAGCAGCAACAT

CACAGGACTGCTGCTGACAAGAGATGGCGGAATCAATGAGAACGGCACCGAAATCTTTAGACCCG

GCGGCGGCGACATGCGGGACAACTGGCGCAGCGAGCTGTACAAGTACAAAGTGGTGAAGATCGA

GCCCCTGGGAGTGGCCCCAACAAAGTGTAAAAGAAGAGTGGTGGGCAGACGGAGACGGAGACGG

GCCGTCGGCATCGGCGCTGTTTTCCTGGGCTTCCTGGGCGCCGCTGGCAGCACCATGGGCGCCGCC

TCTATGACCCTGACCGTTCAGGCTAGACAGCTGCTGAGCGGCATCGTGCAGCAGCAAAACAATTG

CCTGAGAGCCCCTGAGTGCCAGCAAAGAATGCTGCAGCTGACCGTCTGGGGCATTAAGCAACTGC

AAGCCAGGGTGCTCGCTGTGGAGAGATACCTGGGCGATCAGCAGCTGCTGGGCATCTGGGGATGT

AGCGGCAAGCTGATCTGCTGCACCGCCGTGCCTTGGAACGCCAGCTGGTCCAACAAGAGCCTGGA

CAGAATCTGGAACAATATGACATGGATGGAATGGGAGCGGGAAATCGACAACTACACCAGTGAA

ATCTACACACTGATCGAAGAAAGCCAAAACCAGCAGGAGAAGAATGAGCAGGAGCTGCTGGAGC

TGG AC, Ga:cG AG ACC; : A4‘. rCT A A GA (SEQ ID NO: 11

Claims

What is claimed is:

1. A recombinant HIV-1 envelope peptide comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 1 (HV 1303395 JRFL SOSIPv6_MCD5_101nQQavi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO:2 (HV1303396 JRFL SOSIPv6_MCD5_cSorta).

2. A composition comprising any one of the envelopes of claim 1 and a carrier, wherein the envelope is a protomer comprised in a trimer.

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

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

5. The composition of claim 4, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

6. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the trimers of claim 2 or 3.

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

8. The composition of claim 7, wherein the nanoparticle comprises multimers of trimers.

9. The composition of claim 7, wherein the nanoparticle comprises 1-8 trimers.

10. A recombinant nucleotide encoding a HIV-1 envelope peptide comprising all the consecutive amino acids immediately following the signal peptide, wherein the recombinant nucleotide is SEQ ID NO: 3 (encodes HV1303395 JRFL SOSIPv6_MCD5_101nQQavi) or SEQ ID NO: 4 (encodes HV1303396 JRFL SOSIPv6_MCD5_cSorta).

11. A composition comprising any one of the envelopes of claim 1 and a carrier, wherein the envelope is a protomer comprised in a trimer.

12. The composition of claim 11, wherein the envelope is comprised in a stable trimer.

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

14. The composition of claim 13, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

15. A composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the trimers of claim 11 or 12.

16. The composition of claim 15, wherein the nanoparticle is a ferritin self-assembling nanoparticle.

17. The composition of claim 16, wherein the nanoparticle comprises multimers of trimers.

18. The composition of claim 16, wherein the nanoparticle comprises 1-8 trimers.

19. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant envelopes of any one of claims 1-5, in an amount sufficient to induce an immune response.

20. The method of claim 19, wherein the composition is administered as a prime.

21. The method of claim 19, wherein the composition is administered as a boost.

22. A nucleic acid encoding any of the recombinant envelopes of any one of claims 1-5.

23. A composition comprising the nucleic acid of claim 22 and a carrier.

24. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising the nucleic acid of claim 22.

25. A method of inducing an immune response comprising administering an immunogenic composition comprising a prime immunogen followed by at least one boost immunogen, wherein the prime immunogen and/or the boost immunogen comprises any one of the recombinant envelopes or recombinant nucleotides of claim 1 or claim 10, in an amount sufficient to induce an immune response.

26. The method of claim 25, wherein the boost immunogen comprises the recombinant envelopes or recombinant nucleotides.

27. The method of claim 25 wherein the boost immunogen comprises or encodes all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 1 (HV1303395 JRFL SOSIPv6_MCD5_101nQQavi) in any suitable form.

28. The method of claim 26. wherein the boost immunogen comprises or encodes all the consecutive amino acids immediately following the signal peptide in SEQ ID NO:2 (HV1303396 JRFL SOSIPv6_MCD5_cSorta) in any suitable form.

29. The method of claim 25, wherein the prime or boost immunogens are administered as a nanoparticle.

30. The method of claim 29, wherein the nanoparticle is a ferritin nanoparticle.

31. The method of claim 25. wherein the prime or boost immunogens are administered as mRNA-LNP formulation.

32. A composition comprising the recombinant nucleotide of claim 10 and a carrier.

33. A recombinant HIV- 1 envelope peptide comprising R98T, L48Y. S27Y and G57R.