Immunogen design targeting the v3-glycan broadly neutralizing antibody BF520 precursor

Abstract

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

Description

Attorney Docket: 293331 L000100.W01DU8590PCTIMMUNOGEN DESIGN TARGETING THE V3-GLYCAN BROADLY NEUTRALIZING ANTIBODY BF520 PRECURSOR

[0001] This International Patent Application claims the benefit of and priority to U.S. Application No. 63 / 695,222, filed September 16, 2024, entitled “Immunogen Design Targeting the V3-Glycan Broadly Neutralizing Antibody BF520 Precursor,” the content of which is hereby incorporated by reference in its entirety.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 XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 8. 2025, is named DU8590PCT Sequence Listing and is 44,975 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 community working on the HIV-1 epidemic. While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients. ART is not routinely available in developing countries.

[0006] Broadly neutralizing antibodies (bnAbs) that develop during natural infection target the glycans proximal to the third variable region (V3-glycan site) of HIV- 1 envelope (Env).Attorney Docket: 293331 LOOOIOO.WOIDU8590PCT

[0007] The glycans in the first variable region (VI -glycan) as well as the VI peptide can shield the V3-glycan epitope from V3-glycan bnAb recognition.SUMMARY OF THE INVENTION

[0008] Eliciting broadly neutralizing antibodies with vaccination against HIV-1 has been challenging. One roadblock to eliciting broadly neutralizing antibodies to HIV-1 is the highly selective reactivity of the naive B cell receptor that evolves into broadly neutralizing HIV-1 antibodies. The germline targeting strategy for HIV-1 vaccine design aims to overcome this roadblock by designing immunogens that bind the computationally inferred precursor antibody that evolved into a known bnAb.

[0009] In certain embodiments, the invention provides compositions and methods for induction of an immune response, for example cross-reactive (broadly) neutralizing (bn) Ab induction. In particular, in some embodiments, the present invention provides methods to design an immunogen that targets the precursor of the BF520 clone called the BF520 UCA with the aim of eliciting similar antibodies with vaccination.

[0010] An immunogen precisely targeting the BF520 UCA is needed because of its distinct angle of approach from other V3-glycan bnAbs.

[0011] In certain aspects, the invention provides a nucleic acid encoding a recombinant protein comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 1 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 2 (HV1303713; JRFL SOSlPv6_E135W_101nQQavi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 3 (HV1301502_K135W_C- SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 4(HV 1301502 KI 35W lOlnQQ Avi, JRFL SOSIPv6 V 1 PNGS KI 35W 1 OlnQQ avi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 5 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 6 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 7 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) orAttorney Docket: 293331 L000100.W01DU8590PCT comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 8 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi). In some embodiments, nucleic acid sequences with 90. 91. 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 1-8 are disclosed.

[0012] In certain aspects the invention provides an amino acid sequence encoding a recombinant protein comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 9 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 10 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 11 (HV1301502 K135W C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 12 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 13 (HV1303936;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 14 (HV1303938; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 15 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 16 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi). In some embodiments, amino acid sequences with 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 9-16 are disclosed.

[0013] 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.

[0014] 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 toAttorney Docket: 293331 L000100.W01DU8590PCTV3 glycan epitope targeting antibodies. In some embodiments, the selection comprises an immunogen comprising SEQ ID NO: 1 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or SEQ ID NO: 2 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi) or SEQ ID NO: 3 (HV1301502 K135W C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or SEQ ID NO: 4 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or SEQ ID NO: 5 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or SEQ ID NO: 6 (HV 1303938; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or SEQ ID NO: 7 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 8 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi).

[0015] In some embodiments, the selection comprises an immunogen comprising SEQ ID NO: 9 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or SEQ ID NO: 10 (HV1303713;JRFL SOSIPv6_E135W_101nQQavi) or SEQ ID NO: 11 (HV1301502_K135W_C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or SEQ ID NO: 12 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or SEQ ID NO: 13 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C- SORTA) or SEQ ID NO: 14 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or SEQ ID NO: 15 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 16 (HV1303713_N406A_N448D; JRFL S0SIPv6 E135W N406A N448D lOlnQQavi).

[0016] In certain embodiments, the disclosed 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 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- glycan lineages, V lV2-glycan lineages of antibodies.Attorney Docket: 2933311.000100.W01DU8590PCT

[0017] 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 V3 glycan antibody lineages.

[0018] 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 W at position 135. 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-41C, 44A-D, 45, 46, 47 A, 49A-B, 50A-D. 51. 52A-B, 53 A. 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.dl 621 .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 SEQ ID NO: 1 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or SEQ ID NO: 2 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi) or SEQ ID NO: 3 (HV1301502 KI35W C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or SEQ ID NO: 4 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or SEQ ID NO: 5 (HV1303936;Attorney Docket: 293331 LOOOIOO.WOIDU8590PCTJRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or SEQ ID NO: 6 (HV1303938; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or SEQ ID NO: 7 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 8 (HV1303713 N406A N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi) and / or amino acids encoding these envelopes: SEQ ID NO: 9 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or SEQ ID NO: 10 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi) or SEQ ID NO: 11 (HV1301502 K135W C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or SEQ ID NO: 12 (HV1301502_K135W_I01nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or SEQ ID NO: 13 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or SEQ ID NO: 14 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or SEQ ID NO: 15 (HV1303928_N406A_N448D; JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 16 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi).

[0019] In some embodiments, the recombinant HIV-1 envelope comprises a tag at the C- terminus 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.

[0020] 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: 17) or “GDIK” (SEQ ID NO: 18) 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.

[0021] In certain embodiments, the inventive designs comprise glycan holes filled with the introduction of new glycosylation sites to prevent the binding of strain-specific antibodiesAttorney Docket: 293331 EOOOIOO.WOIDU8590PCT that could hinder broad neutralizing antibody development (Wagh, Kshitij et al. “Completeness of HIV-1 Envelope Glycan Shield at Transmission Determines Neutralization Breadth.” 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. 1 1,5 el004932. 29 May. 2015, doi: 10.1371 / joumal.ppat. l004932).

[0022] 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 V 1 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 W at positions 135 (HXB2 numbering).

[0023] In certain embodiments the envelope is a protomer which could be comprised in a stable trimer.

[0024] 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 Fl 4. In other embodiments, the selected mutations are VT8. In certain embodiments, the selected mutations are F14 and VT8 combined.

[0025] 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 16. 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.

[0026] 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.Attorney Docket: 2933311.000100.W01DU8590PCT

[0027] 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.

[0028] In certain embodiments, the invention provides a composition comprising any one of the inventive envelopes, e.g., as disclosed in Table 1 and Figure 16, 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 nanoparticle (LNP).

[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 and Figure 16.

[0030] 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.

[0031] In certain aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises trimers of any of the recombinant HIV-1 envelopes, e.g. as disclosed in Table 1 and Figure 16. In certain embodiments, the nanoparticle is a ferritin self-assembling nanoparticle. In certain embodiments, the nanoparticle comprises multimers of trimers. Provided also are methods for using these compositions comprising nanoparticles.

[0032] 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.

[0033] 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 and Figure 16. In non-limiting embodiments, the envelopes / trimers of the invention are multimeric when comprised in aAttorney Docket: 293331 LOOOIOO.WOIDU8590PCT nanoparticle. The nanoparticle size is suitable for delivery. In non-liming embodiments the nanoparticles are ferritin-based nanoparticles.

[0034] In certain aspects, the invention provides nucleic acids comprising sequences encoding proteins of the invention, e.g., as disclosed in Table 1 and Figure 16. 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.

[0035] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive HIV-1 envelopes, e.g., as disclosed in Table 1 and Figure 16. 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.

[0036] 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.

[0037] 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.

[0038] 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 and Figure 16. 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 ofthe 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.Attorney Docket: 293331 L000100.W01DU8590PCT

[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 any HIV-1 envelope comprising a modifications to the VI loop at positions 134-138 (HXB2 numbering). In certain embodiments, the modification to the VI loop at positions 135 is W.

[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 BF520 UCA neutralization of JRFL pseudotyped virus lacking three glycosylation sites in the first variable region. Neutralization titers are the concentration in pg / mL that inhibits 50% of virus replication (IC50). MuLV is a non-HIV negative control.

[0044] Figure 2 shows BF520 UCA binding to JRFL protein is dependent on JRFL VI region sequence as well as the lack of glycans. JRFL _SOSIPv6 is a recombinant envelope with the natural glycosylated JRFL VI region. JRFL deltaVl PNGS is a recombinant envelope with the natural JRFL VI region lacking three glycosylation sites. JRFL deltaVl PNGS_D13O5V1 is a recombinant envelope with the V 1 region of JRFL replaced by the V 1 region of HIV-1 isolate CH848.D1305. 10.19. The VI region of HIV-1 isolate CH848.D1305.10.19 naturally lacks glycosylation sites.

[0045] Figure 3 illustrates that BF520 UCA has micromolar binding affinity for JRFL deltaVl PNGS. BF520. 1 is the bnAb that is inferred to have evolved from BF520 UCA.

[0046] Figures 4A-B shows superpositions of known structures of V3-glycan bnAbs and shows BF520 UCA has a distinct angle of approach to envelope that does not change with affinity maturation. (A) Comparison between BF520 and other known V3-glycan bnAbs. (B) Comparison of BF520 UCA and BF520.1. JRFL deltaVl PNGS was used as the antigen for both BF520 UCA and BF520.1 structures.

[0047] Figure 5 shows mammalian cell display of JRFL Env variants identify a population of cells that bind to BF520 UCA. Each plot shows fluorescence activated cellAttorney Docket: 293331 L000100.W01DU8590PCT sorting of cells that bound to BF520 UCA. Cells shown on the right were sequenced to identify mutations in envelope that improve BF520 UCA binding.

[0048] Figure 6 shows selected JRFL envelope with N 135 W substitution binds with higher magnitude than wildtype JRFL or JRFL deltaVl PNGS.

[0049] Figure 7 depicts BF520 UCA has higher binding affinity for JRFL N135W than JRFL deltaVl PNGS.

[0050] Figures 8A-8C depict that JRFL N135W binds to the unmutated common ancestor (UCA) and bnAb from the BF520 lineage with the same binding mode. 8 A. 3D reconstruction from negative stain electron microscopy (NSEM) images of three BF520 UCA Fabs bound to engineered envelope JRFL N135W. 8B. 3D reconstruction from negative stain electron microscopy (NSEM) images of three BF520.1Fabs bound to engineered envelope JRFL N135W. 8C. Superposition of the UCA and BF520.1 Fabs bound to JRFL N135W envelope.

[0051] Figure 9 shows adding N135W to JRFL deltaVl PNGS increases binding to BF520 UCA beyond N135W alone.

[0052] Figure 10 shows the removal of N406 and N448 glycans did not improve broadly neutralizing antibody precursor BF520 UCA binding. ELISA binding by serial dilutions of BF520 UCA IgG to JRFL envelopes with VI loop changes (MCD5, VWATN. and E135W) and V4 glycan modifications (N406 and N448 glycans).

[0053] Figures 11A-11B depict nanoparticles displaying JRFL N135W or JRFL N135W with the VI glycans removed binds to the precursor of the DH270 bnAB lineage, but soluble trimer binding is undetectable. Biolayer interferometry binding curves for soluble gpl40 trimers and nanoparticles bearing gpl40 envelope trimers. Each curve shows binding at different envelope or envelope nanoparticle concentrations.

[0054] Figure 12 depicts JRFL N 135 W Env trimer nanoparticle vaccinated macaques generate plasma IgG that binds higher to JRFL N 135 W compared to wildtype JRFL. Symbols are group mean (n = 7). Error bars represent standard deviation.

[0055] Figure 13 shows two of seven macaques show increased binding to envelopes with N 135 W compared to envelope without N135W substitution. ELISA plasma IgG binding titers as log AUC over time. Macaques were immunized twice with envelope nanoparticles and twice with glycopeptide nanoparticles (Man9V3 or glycoV4). B017 and B077 have higher binding titers to JRFL 135W and JRFL deltaVl glycans plus 135W thanAttorney Docket: 293331 L000100.W01DU8590PCTWT or delta VI glycans versions of JRFL. For B077, IgG binding titers to various JRFL envelopes was boosted by glycopeptide nanoparticle immunization.

[0056] Figure 14 shows that Plasma antibody epitope mapping by negative stain electron microscopy (NSEM) shows the presence of VlV3-glycan antibodies. Plasma IgG was digested into Fab fragments, complexed with JRFL N 135 W and visualized by negative stain electron microscopy. 3D reconstructions from NSEM images are shown from a representative monkey.

[0057] Figure 15 depicts that vaccination induces serum neutralization against CON-S and JRFL viruses. Serum was collected two weeks after the fourth immunization. Macaques were immunized with CON-S lacking VI glycans and JRFL N135W envelope nanoparticles two times and two different glycopeptide nanoparticles. Each symbol represents a macaque. Each bar shows group geometric mean. Limit of quantification is shown by gray area.

[0058] Figures 16A-16F show the DNA and amino acid sequences of the immunogens of the present invention.DETAILED DESCRIPTION

[0059] 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.

[0060] 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.

[0061] 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 vims for elucidating the Env evolution pathways that lead to bnAb induction (Nature 496: 469, 2013).

[0062] 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 various combinations of primes and boosts, either as nucleicAttorney Docket: 2933311.000100.W01DU8590PCT 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.

[0063] 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.

[0064] 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).

[0065] 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 Pub20180028645 Al .

[0066] 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.

[0067] 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.

[0068] 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 codonAttorney Docket: 2933311.000100.W01DU8590PCT 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.

[0069] 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.

[0070] The envelope used in the compositions and methods of the invention can be a gpl60, gpl50, gp!45, gp!40, gp!20, 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.

[0071] 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 gp!50. In certain embodiments, gp!40 is designed to form a stable trimer. See z and Figure 16 for non-limiting examples of sequence designs. In certain embodiments envelope protomers form a trimer which is not a SOSIP trimer. In certain embodiment the trimer is a SOSIP based trimer wherein each protomer comprises additional modifications. InAttorney Docket: 293331 LOOOIOO.WOIDU8590PCT 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.

[0072] 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, gp!40, gp!45, gp!50, or gpl60.

[0073] 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.

[0074] 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.

[0075] In certain aspects, the invention provides a cell comprising a nucleic acid encoding any one of the envelopes of the invention suitable for recombinant expression. InAttorney Docket: 2933311.000100.W01DU8590PCT 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.

[0076] 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 acids of such protomers are disclosed herein.

[0077] 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.

[0078] Described herein are nucleic and amino acids 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 gp!60s. In certain embodiments, the described HIV-1 envelope sequences are gpl20s. Other sequences, for example but not limited to stable SOSIP trimer designs, gpl45s, gp!40s, 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), gp!40 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), gp!50s, gp41s, can be readily derived from the nucleic acid and amino acid gp!60 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.

[0079] An HIV-1 envelope has various structurally defined fragments / forms: gpl60; gp 140— including cleaved gpl40 and uncleaved gpl40 (gpl40C), gpl40CF, or gpl40CFI; gp!20 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 ofAttorney Docket: 293331 LOOOIOO.WOIDU8590PCT 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 know n and characterized in the art.

[0080] 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: 19). 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).

[0081] The role of the furin cleavage site was w ell understood both in terms of improving cleavage efficiency, see Binley et al. supra, and eliminating cleavage, see Bosch and Paw-lita, 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).

[0082] 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), gpl40CF and gpl40CFI forms. Envelope gpl40 forms are designed by introducing a stop codon within the gp41 sequence. See Chakrabarti et al. at Figure 1.

[0083] 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 gpl 40C form, two of the R residues in and near the furin cleavage site are changed to E, e.g., RRVVEREKR (SEQ ID NO: 20) is changed to ERVVEREKE (SEQ ID NO: 21), and is one example of an uncleaved gpl40 form. Another example is the gpl40C form which has the REKR site (SEQ ID NO: 19) changed to SEKS (SEQ ID NO: 22). See supra for references.

[0084] 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) atAttorney Docket: 2933311.000100.W01DU8590PCT 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.

[0085] 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: MRVMGIQRNYPQWWIWSMLGFWMLMICNGM Z nGVPVWKEAKTTLFCASDA KAYEKEVHNVWATHACVPTDPNPQE... (SEQ ID NO: 23) (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 gp!40, without an N-terminal Herpes Simplex gD tag substituted for amino acids of the N-terminus of gp!20, 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 entirety7.

[0086] 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.

[0087] 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. InAttorney Docket: 2933311.000100.W01DU8590PCT 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. 7,951,377. In some embodiments the mosaic genes are bivalent mosaics. In some embodiments the mosaic genes are trivalent. In some embodiments, the mosaic genes are administered in a suitable vector with each immunization with Env gene inserts in a suitable vector and / or as a protein. In some embodiments, the mosaic genes, for example as bivalent mosaic Gag group M consensus genes, are administered in a suitable vector, for example but not limited to HSV2, w ould be administered with each immunization with Env gene inserts in a suitable vector, for example but not limited to HSV-2.

[0088] 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.

[0089] 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 responses, both T-cell and humoral responses, are known in the art. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments, DNA is formulated for delivery by a gene gun. In certain embodiments, DNA is administered by electroporation, or by a needle-free injection 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. In certain embodiments DNA is optimized, e.g. codon optimized,Attorney Docket: 2933311.000100.W01DU8590PCT 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, 2011 nov 9.) and nonreplicating (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.

[0090] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies may contemplate using DNA or RNA. or may use complexes of nucleic acid molecules and other entities in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles having 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 Biology, 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.

[0091] 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.

[0092] 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.Attorney Docket: 2933311.000100.W01DU8590PCT

[0093] 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.

[0094] 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 20180028645 Al, US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261172. 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.

[0095] mRNAs delivered in LNP formulations can have advantages over non-LNP formulations. See US Pub 20180028645 Al, US Pub 20190274968, US Pub 20180303925, wherein each content is incorporated by reference in its entirety.

[0096] 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.

[0097] 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.Attorney Docket: 2933311.000100.W01DU8590PCT

[0098] 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.

[0099] 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 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.

[0100] In some embodiments, an 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.

[0101] 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).

[0102] Methods for in vitro transfection of mRNA and detection of envelope expression are known in the art.Attorney Docket: 293331 L000100.W01DU8590PCT

[0103] Methods for expression and immunogenicity determination of nucleic acid encoded envelopes are known in the art.

[0104] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins, including trimers such as but not limited to SOSIP based trimers, suitable for use in immunization are known in the art. In certain embodiments recombinant proteins are produced in CHO cells.

[0105] Envelope glycoproteins referenced in various examples and figures comprise a signal / leader sequence. It is well known 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 gp!20 by homologous and heterologous signal sequences. Virology 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: 24). 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.

[0106] 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).

[0107] 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 doses can range from a few pg micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide.Attorney Docket: 2933311.000100.W01DU8590PCT

[0108] 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.

[0109] 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.

[0110] 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. InAttorney Docket: 2933311.000100.W01DU8590PCT certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different.[OHl] Multimeric Envelopes

[0112] 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 avidity effect that can overcome the low affinity between the antigen and B cell receptor. The initial B cell receptor specific for pathogens can be low affinity, which precludes vaccines from being able to stimulate and expand B cells of interest. In particular, very few 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 / ncommsl2041; Bamrungsap et al. Nanomedicine, 2012, 7 (8), 1253-1271.

[0113] For development as a vaccine immunogen, multimeric nanoparticles that comprise and / or display HIV envelope protein or fragments on their surface can be used.

[0114] 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.

[0115] 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 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. Self-assembling complexes comprising multiple copies of an antigen are one strategy 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). Self-assembling influenzaAttorney Docket: 293331 EOOOIOO.WOIDU8590PCT 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).

[0116] 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 selfassembling 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.

[0117] 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.

[0118] 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: 25). 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: 26), where X signifies any amino acid but most commonly Ala, Ser, Glu.

[0119] 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 envelopeAttorney Docket: 293331 EOOOIOO.WOIDU8590PCT 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.

[0120] Any suitable ferritin sequence could be used. In non-limiting embodiments, ferritin sequences are disclosed in US2019 / 0330279, the content of which is hereby incorporated by reference in its entirety.

[0121] 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 used, so long as the fusion protein is expressed and the trimer is formed.

[0122] 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: 25) 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.Attorney Docket: 293331 LOOOIOO.WOIDU8590PCT

[0123] 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, Current Opinion in Chemical Biology. Volume 22, October 2014. Pages 122-128, ISSN 1367-5931, dx.doi.org / 10.1016 / j.cbpa.2014.09.020; Tabata et al. Anticancer Res. 2015 Aug;35(8):441 1- 7; Pritz et al. J. Org. Chem. 2007, 72, 3909-3912.

[0124] The lipid modified envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0125] The lipid modified and multimerized envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0126] 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.

[0127] Additional 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: 25), 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: 26). where X signifies any amino acid but most commonly Ala, Ser, Glu.

[0128] Table 1, below, shows a summary of envelope sequences disclosed herein and depicted in Figure 16.Attorney Docket: 2933311.000100.W01DU8590PCT

[0129] Table 2, below, 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 W at position 135 (HXB2 numbering).Attorney Docket: 2933311.000100.W01DU8590PCT

[0130] 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).

[0131] 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).Attorney Docket: 2933311.000100.W01DU8590PCT

[0132] V3 glycan Abs bind GDIR (SEQ ID NO: 17). In some embodiments, a change #3 to “GDIR” is needed (e.g. “GDIR” sequence in row 5 of Table 2, supra).

[0133] GDIR / K motif: 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.D0949.10.17 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 critical 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 would 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 with 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.

[0134] 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.Attorney Docket: 2933311.000100.W01DU8590PCT

[0135] 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.

[0136] Any suitable signal peptide could be used. In designs comprising ferritin for multimerization, any suitable linker could be used between the envelope sequence and a ferritin sequence.Attorney Docket: 293331 LOOOIOO.WOIDU8590PCTEXAMPLES

[0137] Example 1

[0138] The broadly neutralizing antibody, BF520 was isolated from a 15-month-old child 11 months post-HIV-1 infection (Simonich et al. Cell. 2016 Jun 30; 166(1): 77-87). The broadest member of the lineage is an antibody called BF520.1, which neutralized 58% of a global panel of viruses (Simonich et al. Cell. 2016 Jun 30; 166(1): 77-87). We sought to develop an immunogen that could bind to the computationally inferred precursor of the BF520 lineage termed BF520 UCA. BF520 UCA share genetic similarities with the precursor for the DH270 lineage, but the pseudotyped virus that is sensitive to DH270 precursor antibody was not sensitive to BF520 UCA (Figure 1). With only five amino acid changes, the DH270 lineage precursor acquires neutralization of the HIV-1 isolate JRFL lacking glycosylation sites in its first variable region (termed JRFL VI N131Q, N137Q, N141Q). We tested BF520 UCA neutralization of this JRFL variant and observed neutralization (Figure 1).

[0139] Example 2

[0140] To determine the importance of the JRFL VI region lacking gly cosylation sites for BF520 UCA binding, we exchanged the JRFL VI region with an aglycosylated V 1 region from CH848.D1305.10.19 virus. Despite lacking VI glycans. the JRFL envelope with the VI region from CH848.D1305. 10.19 virus did not bind to BF520 UCA, whereas the JRFL lacking the three VI glycosylation sites (called JRFL deltaVl PNGS) bound to BF520 UCA (Figure 2).

[0141] Example 3

[0142] The BF520 UCA bound to JRFL deltaVl PNGS with 1.4 LIM affinity. As the antibody acquired somatic mutations the affinity for JRFL deltaVl PNGS improved three orders of magnitude due to a large slowing down of its dissociation rate. The binding was sufficiently strong between the BF520 UCA and JRFL deltaV 1 PNGS for structures of the complex to be discerned. Since V3 gly can-directed antibodies have unique binding modes we sought to determine the binding mode of BF520 UCA. The antigen binding fragment of BF520 UCA bound to JRFL deltaVl PNGS was determined with negative stain electron microscopy (Figure 3). When compared to other known V3 glycan antibodies. BF520 UCA bound to envelope with a unique angle of approach. We also determined the structure ofAttorney Docket: 293331 L000100.W01DU8590PCTBF520.1 and found that affinity maturation did not change the angle of approach set by BF520 UCA (Figure 4).

[0143] Example 4

[0144] Previous studies have suggested that high affinity is preferable for targeting rare bnAb precursors. Thus, we sought to improve the binding affinity between BF520 UCA and JRFL deltaVl PNGS. We designed a I i bran of envelope variants where we mutated the V3- glycan site on JRFL envelope targeted by the BF520 UCA. We either mutated each amino acid in the site one position at a time to all possible amino acids, or we mutated six amino acids at a time in various positions in the V3-glycan site. The variant envelopes were appended to a c-myc tag and an HRV3C cleavage site. Finally, the transmembrane domain for platelet derived grown factor was added c-terminal to the HRV3C cleavage site. 293 T cells were transfected with both envelope libraries.

[0145] Expression of the envelope on the cell surface was confirmed with an antibody against c-myc. Env expressing cells were incubated with BF520 UCA and cells that were positive for BF520 UCA were sorted and put into culture (Figure 5). As a negative control, the wildtype JRFL sequence was examined and showed no binding to BF520 UCA. After two days, hygromycin was added to the cell culture to kill any cells that lacked the envelope library plasmid. The cells were then sorted three more times to enrich a population of high binding cells. The envelope gene in the final sorted cells was sequenced to identify the key envelope mutations that confer better binding to BF520 UCA (Figure 5). The only substitution observed in more than 60% of the sequences was at position 135 in the JRFL VI region. More specifically, 99. 1% of sequences had changed to encode aN135W substitution.

[0146] Example 5

[0147] A recombinant envelope JRFL gpl40 trimer with the N135W substitution was generated to compare different versions of JRFL binding to BF520 UCA. The BF520 UCA bound to JRFL N 135 W with a log area-under-the curve of 9 versus 4.2 for JRFL deltaV 1 PNGS (Figure 6). As a positive control BF520. 1 was tested and bound to both JRFL variant envelopes well (Figure 6). In biolayer interferometry assays, BF520 UCA exhibited a 6-gold improvement in binding affinity compared to JRFL deltaVl PNGS (Figure 7). Thus, the directed evolution of the envelope generated an envelope that bound to a higher magnitude than our initial BF520 UCA-reactive envelope. Lastly, we introduced the N135W substitutionAttorney Docket: 293331 L000100.W01DU8590PCT into JRFL deltaVl PNGS. This envelope bound with the highest magnitude to BF520 UCA (Figure 9).

[0148] Example 6

[0149] To assess the immunogenicity of the JRFL N135W envelope, we conjugated it to the surface of a ferritin nanoparticle with sortase A. We immunized rhesus macaques with the nanoparticle and generated plasma IgG that bound to the JRFL 135W higher than wildtype JRFL. The difference in IgG binding indicates the elicitation of antibodies that are dependent on N135W (Figure 12).

[0150] The envelopes in Table 1 can be produced under cGMP conditions as a recombinant protein and / or mRNA formulated in LNP for use in a Phase I clinical trial.

Claims

Attorney Docket: 293331 LOOOIOO.WOIDU8590PCTWhat is claimed is:

1. A recombinant HIV-1 envelope comprising all the consecutive amino acids immediately following the signal peptide in SEQ ID NO: 9 (HV1303928; JRFL SOSIPv6_E135W_cSorta), in SEQ ID NO: 10 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi), in SEQ ID NO: 11 (HVI301502_K135W_C- SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA), in SEQ ID NO: 12 (HV 13015 O2_K135 W_1 OlnQQ Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi). in SEQ ID NO: 13 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA), in SEQ ID NO: 14 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi), in SEQ ID NO: 15 (HV1303928_N406A_N448D;JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or in SEQ ID NO: 16 (HV1303713_N406A_N448D; JRFL SOSlPv6_E135W_N406A_N448D_101nQQavi).

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 claims 2.

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 method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant envelopes of the preceding claims or compositions of the preceding claims, in an amount sufficient to induce an immune response.Attorney Docket: 293331 L000100.W01DU8590PCT11. The method of claim 10, wherein the composition is administered as a prime.

12. The method of claim 10, wherein the composition is administered as a boost.

13. A nucleic acid encoding any of the recombinant envelopes of the preceding claims.

14. A composition comprising the nucleic acid of claim 13 and a carrier.

15. The composition of claim 14, wherein the nucleic acid comprises a sequence encoding all the consecutive amino acids immediately following the signal peptide encoded in: SEQ ID NO: 1 (HV1303928; JRFL SOSIPv6_E135W_cSorta), SEQ ID NO: 2 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi), SEQ ID NO: 3(HV 1301502 K135 W_C-SORTA; JRFL_SOSIPv6_V 1_PNGS_K135 W_C-SORTA), SEQ ID NO: 4 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi), SEQ ID NO: 5 (HV1303936; JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA), SEQ ID NO: 6 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi). SEQ ID NO: 7 (HV1303928_N406A_N448D;JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 8 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi).

16. A method of inducing an immune response in a subject comprising administering an immunogenic composition comprising the composition of claim 15.

17. A method of inducing an immune response comprising administering an immunogenic composition comprising a prime immunogen followed by at least one boost immunogen comprising any one of the recombinant envelopes of either claim 1 , or claim 15, in an amount sufficient to induce an immune response.

18. The method of claim 17, wherein a first boost immunogen comprises any one of the recombinant envelopes of either claim 1, or claim 15.

19. The method of claim 16, wherein the boost immunogen comprises a sequence encoding all the consecutive amino acids immediately following the signal peptide encoded in SEQ ID NO: 1 (HV1303928; JRFL SOSIPv6_E135W_cSorta) or SEQ ID NO: 2 (HV1303713; JRFL SOSIPv6_E135W_101nQQavi) or SEQ ID NO: 3 (HV1301502 K135W C-SORTA; JRFL_SOSIPv6_Vl_PNGS_K135W_C-SORTA) or SEQ ID NO: 4 (HV1301502_K135W_101nQQ_Avi, JRFL_SOSIPv6_Vl_PNGS_K135W_101nQQ_avi) or SEQ ID NO: 5 (HV1303936;Attorney Docket: 2933311.000100.W01DU8590PCTJRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_C-SORTA) or SEQ ID NO: 6 (HV1303938;JRFL_SOSIPv6_Vl_PNGS_K135W_N406A_N448D_101nQQ-avi) or SEQ ID NO: 7 (HV1303928_N406A_N448D;JRFL_SOSIPv6_E135W_N406A_N448D_cSorta) or SEQ ID NO: 8 (HV1303713_N406A_N448D; JRFL SOSIPv6_E135W_N406A_N448D_101nQQavi) in any suitable form.

20. The method of any one of claims 16-19, wherein the prime or boost immunogen are administered as a nanoparticle.

21. The method of claim 20. wherein the nanoparticle is a ferritin nanoparticle.

22. The method of any one of claims 16-19, wherein the prime or boost immunogen are administered as mRNA-LNP formulation.

Images