Membrane proximal external region (MPER) from HIV that stimulates broadly neutralizing antibodies (BNABS)

Abstract

Disclosed are membrane proximal external region (MPER) immunogens from HIV that can stimulate broadly neutralizing antibodies (bnABS), lipid immunogen displays having the MPER immunogens, and methods for stimulating antibodies using the MPER immunogens and lipid immunogen displays.

Description

MEMBRANE PROXIMAL EXTERNAL REGION (MPER) FROM HIV THAT STIMULATES BROADLY NEUTRALIZING ANTIBODIES IbnAbs)

[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 540,482, filed on September 26, 2023, the entire contents of which are incorporated herein by reference.GOVERNMENT INTERESTS

[0004] This invention was made with government support from the NIH, NIAID, Division of AIDS for UM1 grant Al 144371. The government has certain rights in the invention.SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on DATE, is named FILE_NAME and is SIZE bytes in size.FIELD

[0006] The present disclosure provides compositions and methods for inducing anti -HIV- 1 antibodies.BACKGROUND

[0007] While anti-retroviral treatment (ART) has dramatically prolonged the lives of HIV- 1 infected patients, ART is not routinely available in developing countries. Development of a safe and effective HIV-1 vaccine is a high priority of the scientific community working on the HIV-1 epidemic.SUMMARY

[0008] Disclosed herein is a membrane proximal external region (MPER) immunogen from human immunodeficiency virus. In some embodiments, the MPER immunogens can comprises an amino acid sequence SEQ ID NO: 1-8, and 14-135 or a sequence at least 90% identical thereto.

[0009] Disclosed herein is a lipid immunogen display comprising an amino acid sequence SEQ ID NO: 1-8, and 14-135 or a sequence at least 90% identical thereto. In some embodiments, the lipid immunogen display can be a liposome.

[0010] Disclosed are methods for stimulating an immune response (e.g., virus neutralizing antibodies) against HIV using the disclosed MPER immunogens or lipid immunogen displays having the disclosed MPER immunogens.BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 shows a schematic depicting an example strategy for prime and boost peptide- liposome immunogen designs targeting both proximal and distal MPER bnAb epitopes. UCA is unmutated common ancestor; IA is intermediate antibody; bnAb is broadly neutralizing antibody.

[0012] FIG. 2A illustrates an example MPER peptide-liposome showing an MPER peptide (red) and a Gag p24 Th-epitope peptide (black) associated with a lipid bi-layer (blue) of the liposomes. The table shows example sequences of the variant MPER656 peptides (MPER656.1, MPER656.2, MPER656.3; SEQ ID NOs: 2, 3 and 4, respectively) with amino acid substitutions marked in color. The relative binding of DH511 bnAB binding is graded based on the difference in the magnitude of the binding response. MPER656 is SEQ ID NO: 1.

[0013] FIG. 2B shows a graph depicting bio-layer interferometry (BLI) binding curves of DH511 bnAb to each of the indicated MPER peptide-liposomes.

[0014] FIG. 2C shows a graph depicting an example of BLI overlay ed curves showing binding of the proximal MPER bnAb 2F5 to each of the MPER656 liposomes.

[0015] FIG. 3 shows amino acid sequences of MPER656.1 peptide-liposome construct variants. MPER656.1 is SEQ ID NO: 2; UCA.OPT (also called Variant 1) is SEQ ID NO: 5; UCA.OPT2 (also called Variant 2) is SEQ ID NO: 6; DUAL.UCA.OPT (also called Variant 3) is SEQ ID NO: 7; DUAL.UCA.OPT2 (also called Variant 4) is SEQ ID NO: 8.

[0016] FIG. 4A shows a graph depicting an example of overlayed BLI curves of DH1317 lineage mAh binding to MPER peptide-liposomes.

[0017] FIG. 4B shows a graph depicting an example of the BLI binding responses (nm) of the indicated DH1317 mAbs to variants of the MPER656.1 -liposomes. Binding affinity (KD, nM) of DH1317 mAb to MPER656-liposomes is given (in red) for each of the indicated mAbs.

[0018] FIG. 5 shows graphs depicting examples of overlayed BLI curves showing binding of DH1317 UCA (FIG. 5A) or the first intermediate IA4 (FIG. 5B) to Variant 4 peptide- liposomes or MPER656.1 liposomes.

[0019] FIG. 6. FIG. 6A shows a graph depicting an example of overlayed BLI curves of DH511 lineage mAh binding to MPER656 peptide-liposomes. FIG. 6B shows a graph depicting an example of BLI binding responses (nm) of each of the indicated DH511 lineage mAbs to variants of the MPER656.1 -liposomes.

[0020] FIG. 7 shows a graph depicting example MPER antibody binding to an mRNA- encoded MPER construct expressed on the cell surface (MPER656 + TMD sequence; MPER.B-TMD Y712I), as discussed in Example 4. Flow cytometric mean fluorescence intensity (MFI) values are plotted for each of the labeled proximal and distal MPER bnAbs or UCAs, as well as the HVTN133 derived MPER mAbs following labeling of cells expressing the encoded MPER.

[0021] FIG. 8 shows a schematic of a representative immunization regimen showing the down-selected Variant 4 peptide-liposomes as the first boost to be followed by the mRNA- encoded membrane-bound MPER.

[0022] FIG. 9 illustrates 2nd Generation MPER peptide prime immunogen variants of the MPER 656 sequence. The sequences of the MPER peptide variants (M1-M5) were selected from a yeast display library for improved binding to DH511UCA.

[0023] FIG. 10 shows DH511 lineage mAbs Binding to 2nd Generation MPER peptide variant peptides and DV500-P-PL peptide for comparison. Biolayer Interferometry (BLI) assay shows binding of DH511 lineage mAbs to variant M1-M4 peptides. Each biotin tagged peptide was anchored to streptavidin coated sensor tips and dipped into wells containing each of the listed mAbs (100 pg / mL).

[0024] FIG. 11 shows DH511 lineage mAh Binding Responses to DV500-P-PL and 2nd Generation MPER peptide variant peptide liposomes. BLI assay showing binding responses of each DH511 lineage mAbs to variant M1-M4 peptide-liposomes and to liposomes without peptide (connecting solid line). Binding responses to the first generation DV-500-P-PL areconnected (dashed line). DV500-P-PL_N671G includes a single ammino substitution (N671G) to overcome a steric clash encountered by early intermediate antibodies. Each biotin tagged peptide was anchored to streptavidin coated sensor tips and dipped into wells containing 100 pg / mL each of the listed mAbs .

[0025] FIG. 12 illustrates DH511 lineage mAbs binding to MPER peptide liposomes. BLI assay showing binding responses of each DH511 lineage mAbs to and 2nd Generation MPER peptide variant M1-M4 peptide-liposomes and to DV500-P-PL (*) with a single amino acid substitution (DV500-P-PL- N671G). Each biotin tagged peptide was anchored to streptavidin coated sensor tips and dipped into wells containing each of the listed mAbs.

[0026] FIG. 13 shows a schematic of env-bNAb coevolution inspired vaccine design strategy: use bNAb intermediate resistance mutations & sequence diversity coverage. For the priming immunogen use Env signatures associated with sensitivity to earliest intermediates. Identify Env resistance signatures involved in: intermediate X -> viral escape -> Ab maturation to intermediate X+l. Design vaccines incorporating these signatures that provide: Affinity gradient tow ards next intermediate; Improved presentation of HIV- 1 sequence diversity (mechanism for heterologous breadth development in infections)

[0027] FIG. 14 shows a schematic of the vaccine strategy for DH511 antibody lineage. Identify Env signatures associated with each group and use these for design of prime & boost immunogens.

[0028] FIG. 15 that signature amino acids show uniform trends with DH511 potency. D-674 enriched with highest sensitivity for DH51 1 and becomes a priming immunogen. Glycans at aa 674 are enriched in high / intermediate sensitivity viruses and can be used for early boosting. S-674 aa enriched in low- sensitivity / resistant viruses and can be used for late boosting.

[0029] FIG. 16 shows date where amino acids E-662 & K-677 lower frequency in the most sensitive viruses but decreasing frequency with increasing titers, so associated with sensitivity. Blue / Light blue= sensitive to early intermediates and a decrease in frequency vs increasing ICso. Green = sensitive to middle / late intermediates and an increase in frequency vs increasing ICso. Red = enriched in resistant viruses.

[0030] FIG. 17 is a table showing target Env signatures for immunogen designs. These are used for proximal and distal UCA targeting, and / or DH511 boosts, These can also be used for proximal DH1317.4 targeting.

[0031] FIG. 18 shows amino acid optimization based on dual UCA reactivity. Blue means DH511 sensitive; green means DH511 breadth of reactivity gain; black is non-significant; and yellow is DH1317 sensitive.

[0032] FIG. 19 is a heatmap showing boost selection from neutralization and sequence data. Boosts chosen from 12 viruses with intermediate neutralization data. Blue=DH511 sensitive; green=breadth gain; black=non-significant. Upper arrow shows 129 sensitivity; middle arrow shows 129-resistant and 128 sensitive; and lower arrow shows 128 resistant and 127 sensitive.

[0033] FIG. 20. FIG. 20A is a table showing selected DH511 boosts, showing the clade and virus, the affinity gradient and the amino acid signatures. FIG. 20B shows example amino acid frequency of signatures where blue indicates the signature is covered by the prime; green indicates the signature is covered by the boosts; and black is not covered.DETAILED DESCRIPTION

[0034] The virion lipid can be an important component of the binding immunogen for the gp41 membrane proximal external region (MPER) neutralizing epitope of HIV. Herein are disclosed immunogens to recreate a neutralizing epitope that includes the gp41 MPER associated with lipid (e.g., liposomes). In some embodiments, the disclosed peptide liposomes bind antibodies that can bind to the distal conserved region of MPER (e.g., antibodies from the DH511 antibody lineage) and the proximal conserved region of MPER (e.g.. 2F5 antibody).DEFINITIONS

[0035] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0036] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0037] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitlystated otherwise. Similarly, “an example;’ “exemplary"’ and the like are understood to be nonlimiting.

[0038] The term '‘substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0039] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has.” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a. b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0040] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).IMMUNIZATION METHODS

[0041] bnAbs are antibodies that can neutralize multiple HIV-1 viral strains, and they can be unique because they can target conserved epitopes of the virus; therefore, the virus may mutate, but the targeted epitopes will still exist.

[0042] A goal of HIV vaccine development is to elicit broadly neutralizing antibodies (bnAbs) against the HIV virus. Although some HIV infected individuals can make bnAbs, high levels of bnAbs are rarely observed upon HIV infection and it can take years for these bnAbs antibodies to develop (Swanson, O., Cell Reports 36, 109561, August 17, 2021).

[0043] During this development from an unmutated common ancestor (UCA) to their mature form, these bnAbs follow a maturation pathway that includes early and late intermediate antibodies (early IAS and late IAS) that can acquire a large number of somatic mutations. Some of the somatic mutations can be “functional mutations” sufficient to provide neutralization breadth and potency, while others are “improbable mutations,” or amino acid changes that are expected to happen with low frequency naturally (Hwang et al., 2017; Yaariet al., 2013). While not wishing to be bound by any theory, since improbable functional mutations generally represent rare events that restrict bnAb development, antibodies with such sequence changes will likely need to be explicitly selected by candidate immunogens in vaccinations.

[0044] The elicitation of bnAbs by an HIV vaccine can involve priming the immune system to activate antibody precursors, followed by boosting immunizations to select for antibodies with functional features required for neutralization breadth. A first inoculation can be termed a “primary inoculation, or prime” and subsequent immunizations can be termed “booster inoculations, or boosts”. Booster inoculations can enhance the immune response, and immunization regimens including at least one booster inoculation can be used. Any composition of the invention can be used for a prime or boost immunization. The immune response against the compositions of the invention can be generated by one or more inoculations of a subject with an immunogenic composition disclosed herein.

[0045] A feature of vaccine design can also be the creation of affinity gradients of the Env immunogens across stages of bnAb B cell lineage development, in order to create an ‘affinity pull’ across the lineage such that a ceiling of affinity is not reached until the B cell lineage achieves full maturation (Haynes, Barton F., et al. "Strategies for HIV-1 vaccines that induce broadly neutralizing antibodies." Nature Reviews Immunology 23.3 (2023): 142-158).

[0046] In some aspects, this disclosure describes strategies that can elicit an immune response against HIV in a subject. In some embodiments, the strategies include priming the immune system to activate antibody precursors, followed by boosting immunizations to select for antibodies with functional features required for neutralization breadth. In some embodiments, the strategies include prime and boost immunogens that include lipids (e.g., lipid immunogen displays); broad neutralizing antibodies that recognized proximal and distal gp41 MPER epitopes; binding of the gp41 MPER epitopes with an affinity gradient to each of the intermediate antibodies in the bnAb maturation pathway; targeting of key functional and improbable mutations in key intermediates, or a combination thereof. In some embodiments, the strategy includes prime and boost immunogens that include lipids. In some embodiments, the strategy includes broad neutralizing antibodies that can recognize proximal and distal gp41 MPER epitopes. In some embodiments, the strategy includes binding of the gp41 MPER epitopes with an affinity gradient to IAS in the bnAb maturation pathway. In some embodiments, the strategy includes targeting of key functional and improbable mutations in certain IAs. In some embodiments, the gp41 MPER epitope can bind with higher affinity to late IAs than to intermediate IAs. In some embodiments, the gp41 MPER epitopecan bind with higher affinity to intermediate IAS than to early IAS. In some embodiments, the gp41 MPER epitope can bind with higher affinity to early IAs than to the UCA.

[0047] In some embodiments, the prime can elicit UCA antibodies. In some embodiments, the boost can elicit early IAs. In some embodiments, the boost can elicit intermediate IAs. In some embodiments, the boost can elicit late IAs. In some embodiments, bnABs can be elicited.NEUTRALIZING ANTIBODIES BINDING TO MPER

[0048] Studies of HIV- 1 -neutralizing antibody co-evolution have identified clonal ‘trees’ of bnAb B cells, their inferred bnAb unmutated common ancestors (UCAs) and the transmitted / founder viruses or their progeny that are bound by the BCRs of lineage members. Several clonal trees of neutralizing antibodies that bind MPER have been described.

[0049] For example, naturally induced monoclonal antibodies 2F5, 4E10, 10E8 isolated from HIV-infected individuals displayed a broadly neutralizing activity’. 2F5 targets the linear sequence 662ELDKWA667 (SEQ ID NO: 9) within the N-terminal moiety of the MPER, also called ‘’proximal” sequence or epitope. 4E10 and 10E8 target the “distal” conserved tryptophan rich moiety' located C-terminal to the 2F5 epitope which includes the sequence 67iNWFDIT676 (SEQ ID NO: 11).

[0050] Other lineages of HIV- 1 -neutralizing antibodies include the DH511 and DH1317 lineages. DH511 is a distal MPER-specific bNAbs that was isolated from plasma of an African HIV-1 clade C chronically infected individual. Six clonally related monoclonal antibodies (mAbs). designated DH511.1 to DH511.6, were isolated by antigen-specific single memory B cell sorting using MPER peptide fluorophore-labeled probes (Williams et al., Sci Immunol. 2017 Jan 27; 2(7): eaal2200.). The DH1317 is a lineage in which the intermediate (I A3) has two key functional and improbable mutations (Williams, W., www.medrxiv.org / content / medrxiv / early / 2023 / 03 / 14 / 2023.03.09.23286943.full.pdl). The DH1317 lineage is a proximal gp41 MPER neutralizing antibody lineage isolated from a HVTN133 clinical trial vaccinee.MPER LIPOSOMES AND PEPTIDES

[0051] The membrane proximal external region (MP ER) is a conserved region within the HIV Env gp41 subunit and, therefore, a vaccine target for vaccine development. MPER peptides alone as immunogens can be ineffective for the induction of bnAbs; however, studies using synthetic peptide-liposome conjugates have shown that membrane anchoring is important for the efficient presentation of MPER epitopes. For example, rhesus macaques immunized using MPER peptides displayed on liposomes developed antibodies that recognized those MPER proximal epitopes, and in humans produced heterologous neutralizing antibodies (HVTN 133 trial; classic.clinicaltrials.gov / ProvidedDocs / 41 / NCT03934541 / Prot_SAP_002.pdf).

[0052] In some aspects, the invention described herein includes MPER peptide-lipid immunogen displays. A lipid immunogen display can be a lipid(s) that can display an immunogen such that an immune response can be stimulated. In some embodiments, the lipid in a lipid immunogen display can be a lipid bilayer. In some embodiments, the lipid in a lipid immunogen display can be a micelle. In some embodiments, the lipid in a lipid immunogen display can be a cell surface membrane. In some embodiments, the lipid in a lipid immunogen display can be a liposome. In some embodiments, the lipid immunogen display can display an immunogen that is a peptide having an MPER region. In some embodiments, the lipid immunogen display can be a MPER liposome.

[0053] In some embodiments, the MPER peptides can be displayed on a surface of a composition that does not have a lipid. In some embodiments, an immunogen display can include a scaffold, nanoparticle, virus-like particle (VLP) or a self-assembling protein nanoparticle. In some embodiments, the immunogen display has a lipid. In some embodiments, the immunogen display does not have a lipid. In some embodiments, the immunogen is encoded by mRNA that expresses the MPER with a transmembrane domain (TMD) in a trimeric form displayed on the membrane surface of a cell.

[0054] The MPER liposome can include an MPER component and a lipid component. In some embodiments, the lipid component can induce membrane binding. In some embodiments, the MPER component can include an MPER-656 peptide sequence (SEQ ID NO 1) and / or variants thereof (SEQ IDs Nos 2-8, 14-135). In some embodiments, the MPER- 656 peptide sequence (SEQ ID NO: 1) can be derived from the membrane proximal region of HIV-1 gp41. In some embodiments, the MPER-656 peptide can include the core epitopes of the broadly neutralizing mAbs 2F5 (SEQ ID NO: 10), 4E10 (SEQ ID NO: 11), DH511 and 10E8 (SEQ ID NO: 12), or a combination thereof.

[0055] In some embodiments, the MPER-656 peptide can be synthesized with a C-terminal hydrophobic membrane anchor tag or Th-epitope (SEQ ID NO: 9) which can facilitate anchoring of the peptide to synthetic liposomes and presentation of the optimal peptide conformation for MPER bnAb binding, and can occlude the binding of non-neutralizing MP ER antibodies. The gp41 MPER-656 peptide liposome immunogen has been shown to be able to induce triggering of anergic 2F5 bnAb cells in 2F5 knock-in mice, resulting in high levels of plasma bnAb. In some embodiments, the MPER-656 peptide and / or variants thereof include a Th-epitope. In some embodiments, the amino acide sequence of the Th-epitope is set forth in SEQ ID NO: 9.

[0056] MPER656 liposomes can bind to 4E10 and 10E8, bnAbs that recognized the distal MPER (PMID: 22140469; 19640992). However, MPER656 liposomes do not bind to DH511 lineage members due to the lack of key residues including D674 that make up the DH511 core epitope. Disclosed herein are MPER peptide-liposomes including MPER derived peptides that can bind to members of the distal MPER bnAb DH511 lineage, e.g., 110, Il 1, DH511,.2, 127. 128, UCA, and 129. In some embodiments, these MPER derived peptide- liposomes that can bind to members of the distal MPER bnAb DH511 lineage can carry an MPER peptide that has that amino acid sequence set forth in SEQ ID NO: 1.

[0057] Some aspects of this invention can describe MPER656 derived MPER peptide- liposomes that can bind to members of the distal MPER bnAb DH511 lineage.

[0058] In some embodiments, the MPER656 derived peptide-liposome that can bind to any member of the distal MPER bnAb DH51 1 lineage can be MPER656. 1 can have the amino acid sequence set forth in SEQ ID NO: 2 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto. In some embodiments, MPER656.1 can bind DH511 bnAb members. In some embodiments. MPER656.1 can bind DH511.2.

[0059] In some embodiments, the MPER656 derived peptide-liposome that can bind to any member of the distal MPER bnAb DH511 lineage can be MPER656.2 and can have the amino acid sequence set forth in SEQ ID NO: 3 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto. In some embodiments, MPER656.2 can bind DH511.

[0060] In some embodiments, the MPER656 derived peptide-liposome that can bind to any member of the distal MPER bnAb DH511 lineage can be MPER656.3 and can have the amino acid sequence set forth in SEQ ID NO: 4 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto. In some embodiments, MPER656.3 can bind DH511.

[0061] In some embodiments, the MPER656 derived peptide-liposome can bind to proximal MPER bnAb 2F5. In some embodiments, MPER656.1 can bind to proximal MPER bnAb2F5. In some embodiments, MPER656.2 can bind to proximal MPER bnAb 2F5. In some embodiments, MPER656. 1 can bind proximal MPER bnAb 2F5 and distal MPER bnAb DH511.2.

[0062] In some aspects, this invention describes peptides whose sequences are variations of the MPER656.1 sequence designed to optimize MPER bnAb epitopes (FIG. 3) for both proximal and distal MPER bnAbs. In some embodiments, the sequence optimization is based on neutralization breadth signatures of the proximal (DH1317.4) and the distal (DH511) MPER bnAbs

[0063] In some embodiments, the peptide with an optimized sequence can be UCA.OPT, and can have a sequence set forth in SEQ ID NO: 5 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0064] In some embodiments, the peptide with an optimized sequence can be UCA.OPT2, and can have a sequence set forth in SEQ ID NO: 6 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0065] In some embodiments, the peptide with an optimized sequence can beDUAL. UCA.OPT. and can have a sequence set forth in SEQ ID NO: 7 or a sequence 90. 91. 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0066] In some embodiments, the peptide with an optimized sequence can beDUAL.UCA OPT2, and can have a sequence set forth in SEQ ID NO: 8 or a sequence 90, 91, 92. 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0067] In some aspects, this invention describes peptides whose sequences are variations of the MPER656.1 sequence designed to optimize MPER bnAb epitopes (FIG. 3) for both proximal and distal MPER bnAbs using yeast display technology. In some embodiments, the sequence optimization is based on neutralization breadth signatures of the distal (DH511) MPER bnAbs.

[0068] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NO: 14 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0069] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NO: 15 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0070] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NO: 16 or a sequence 90. 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0071] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NO: 17 or a sequence 90. 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0072] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NO: 18 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.

[0073] In some embodiments, the peptide with an optimized sequence can be a 2ndGeneration variant, and can have a sequence set forth in SEQ ID NOS: 19-135 or a sequence 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto as set forth in Table 1.

[0074] In some aspects, this invention describes MPER peptide-liposomes including any of the peptides whose sequences are variations of the MPER656.1 sequence designed to optimize MPER bnAb epitopes shown in FIG. 3. In some embodiments, the MPER peptide- liposome with an optimized sequence can be Variant 1 and includes peptide UCA.OPT. In some embodiments, the MPER peptide-liposome with an optimized sequence can be Variant 2 and includes peptide UCA.OPT2. In some embodiments, the MPER peptide-liposome with an optimized sequence can be Variant 3 and includes peptide DUAL. UCA.OPT. In some embodiments, MPER the peptide-liposome with an optimized sequence can be Variant 4 and includes peptide DUAL.UCA.OPT2.

[0075] In some aspects of this invention, any of the MPER peptide-liposomes including any of the peptides whose sequences are variations of the MPER656. 1 sequence designed to optimize MPER bnAb epitopes shown in FIG. 3 can bind to any members of the DH1317 lineage of mAbs (e.g., DH1317.4, IA2, IA3, IA4, and UCA). In some embodiments, Variant 1 can bind to any members of the DH1317 lineage of mAbs. In some embodiments, Variant 2 can bind to any members of the DH1317 lineage of mAbs. In some embodiments, Variant 3 can bind to any members of the DH1317 lineage of mAbs. In some embodiments, Variant 4 can bind to any members of the DH1317 lineage of mAbs.

[0076] In some embodiments, Variant 4 can bind to DH1317 UCA. In some embodiments, Variant 4 can bind to DH1317 first intermetidate 4 (IA4). In some embodiments, Variant 4 can bind to DH1317UCA can be enhanced when compared with binding of the MPER656.1 peptide-liposome to DH1317UCA. In some embodiments, DH1317 IA4, the UCA-proximal intermediate, can include two functional and improbable mutations. In some embodiments, DH1317 IA4, the UCA-proximal intermediate, can be a key intermediate for a vaccine target.

[0077] In some aspects of this invention, any of the MPER peptide-liposomes including any of the peptides whose sequences are variations of the MPER656. 1 sequence designed tooptimize MPER bnAb epitopes show n in FIG. 3 can bind to any members of the DH11 lineage of mAbs (e.g., 110. Il l, DH511,.2, 127, 128. UCA, and 129). In some embodiments, Variant 1 can bind to any members of the DH1317 lineage of mAbs. In some embodiments,Variant 2 can bind to any members of the DH1317 lineage of mAbs. In some embodiments,Variant 3 can bind to any members of the DH1317 lineage of mAbs. In some embodiments,Variant 4 can bind to any members of the DH1317 lineage of mAbs.

[0078] In some embodiments. Variant 4 can bind to any of the affinity mature DH51 1 IAS. In some embodiments, Variant 4 can bind to DH511.2. In some embodiments. Variant 4 binding to any of the affinity mature DH511 IAs can be enhanced when compared with binding of the MPER656. 1 peptide-liposome to any of the affinity mature DH511 IAs. In some embodiments, Variant 4 binding to any of the affinity mature DH511.2 can be enhanced when compared with binding of the MPER656. 1 peptide-liposome to DH511.2.

[0079] In some embodiments, Variant 4 can bind with increasing strength to both the proximal and distal gp41 MPER bnAbs.

[0080] In some embodiments, binding of Variant 4 to the DH1317 intermediate (I A3) can be increased when compared to binding MPER656.1 liposomes to the DH1317 intermediate (IA3).

[0081] In some embodiments, binding of the variant 4 MPER liposomes show ed enhanced binding to IAs of the DH511 bnAb lineage.

[0082] In some aspects, this invention describes MPER peptide-liposomes including any of the peptides whose sequences are 2nd Generation variations of the MPER656.1 sequence designed to optimize MPER bnAb epitopes shown in FIG. 10. In some embodiments, the MPER peptide-liposome with an optimized sequence can be Ml. In some embodiments, the MPER peptide-liposome with an optimized sequence can be M2. In some embodiments, the MPER peptide-liposome with an optimized sequence can be M3. In some embodiments, MPER the peptide-liposome with an optimized sequence can be M4. In some embodiments, MPER the peptide-liposome with an optimized sequence can be M5.

[0083] In some aspects, this invention describes MPER peptide-liposomes including any of the peptides whose sequences are 2nd Generation variations of the MPER656. 1 sequence designed to optimize MPER bnAb epitopes shown in FIG. 10 can bind to any members of the affinity mature DH511 lineage of mAbs (e.g., DH511.2, d96, 127, 128, 129 and UCA). In some embodiments, Ml can bind to any members of the DH511 lineage of mAbs. In some embodiments, M2 can bind to any members of the DH511 lineage of mAbs. In some embodiments, M3 can bind to any members of the DH511 lineage of mAbs. In someembodiments, M4 can bind to any members of the DH511 lineage of mAbs. In some embodiments, M5 can bind to any members of the DH511 lineage of mAbs.

[0084] In some embodiments, any of the MPER peptide-liposomes including any of the MPER peptides set forth in any of SEQ IDs NO: 1-8 and 14-135 can be displayed on the surface of a cell as an mRNA-encoded MPER peptide. In some embodiments, any of the mRNA-encoded peptides can bind to any of the antibodies described herein. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of the proximal bnAbs described herein. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of the distal bnAbs described herein. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of MPER bnAbs and bnAb UCAs DH511. 2F5 RUA D, 2F5 RUA N, 2F5 IgGl, Ab 10E8, Ab 4E10, DH570. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of HVTN133 MPER mAbs DH1355, DH1318.2, DH1347.2, DH1348, DH1317.8, DH1346, DH1322.1. DH1315.3, DH1316.2, DH1316.10, DH1321.1, DH1317.9, and DH1317.4 (for HVTN133, see classic.clinicaltrials.gov / ProvidedDocs / 41 / NCT03934541 / Prot_SAP_002.pdf).

[0085] In some embodiments, any of the MPER peptide-liposomes including any of the MPER peptides set forth in any of SEQ IDs NO: 14-135 can be displayed on the surface of a cell as an mRNA-encoded MPER peptide. In some embodiments, any of the mRNA-encoded peptides can bind to any of the antibodies described herein. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of the proximal bnAbs described herein. In some embodiments, any of the mRNA-encoded peptides described herein can bind to any of the distal bnAbs described herein. In some embodiments, any of the mRNA- encoded peptides described herein can bind to any of MPER bnAbs and bnAb UCAs affinity mature DH511 lineage of mAbs (e.g., DH511.2, d96, 127, 128, 129 and UCA).

[0086] Some aspects of this invention describe an immunization regimen based on a primeboost design for eliciting an immune response to HIV. In some embodiments, the regimen can be used to immunize subj ects against HIV infection. In some embodiments, the design can include applying to a subject a prime including any one of Variant 1. Variant 2, Variant 3 and / or Variant 4 liposomes, and / or any one of the MPER derived peptide-liposomes including any of peptides with any of the sequences set forth in SEQ ID NOs: 1-4, and / or a MPER / TMD (mRNA-encoded) including any of the peptides with sequences set forth in SEQ IDs NO: 1-8); and one or more boosts including any one of Variant 1, Variant 2, Variant 3 and / or Variant 4 liposomes, and / or any one of the MPER peptide-liposomes including any ofpeptides with any of the sequences set forth in SEQ ID NOs: 1-4; and / or a MPER / TMD (mRNA-encoded) including any of the peptides with sequences set forth in SEQ IDs NO: 1-8 and SEQ ID NOST4-135.

[0087] In some embodiments, the immunization regimen can be used to immunize subjects against HIV infection. In some embodiments, the design can include applying to a subject a prime including a Variant 1 liposome that can elicit an immune response including UCAs; a boost including a Variant 2 liposome that can elicit an immune response including early IAS; a boost including an MPER656. 1 peptide-liposome that can elicit an immune response including late IAs; and / or a MPER / TMD (mRNA-encoded) that can elicit an immune response including late lA / bnAbs.

[0088] In some embodiments, the immunization regime can include applying to a subject a prime including a liposome carrying the MPER656. 1 peptide (SEQ ID NO: 2) that can elicit an immune response including UCAs; a boost including a variant 4 liposome (SEQ ID NO: 8) that can elicit an immune response including early IAs and late IAs; and a boost including an MPER / TMD (mRNA-encoded) that can elicit an immune response including late IA and / or bnAbs.

[0089] Some aspects of this invention describe an immunization regimen based on a primeboost design for eliciting an immune response to HIV. In some embodiments, the regimen can be used to immunize subjects against HIV infection. In some embodiments, the design can include applying to a subject a prime including any one of Ml, M2, M3. M4 and / or M5 liposomes, and / or any one of the MPER derived peptide-liposomes including any of peptides with any of the sequences set forth in SEQ ID NOs: 14-135, and / or a MPER / TMD (mRNA- encoded) including any of the peptides with sequences set forth in SEQ IDs NO: 14-135); and one or more boosts including any one of Ml, M2. M3, M4 and / or M5 liposomes, and / or any one of the MPER peptide-liposomes including any of peptides with any of the sequences set forth in SEQ ID NOs: 14-135; and / or a MPER / TMD (mRNA-encoded) including any of the peptides with sequences set forth in SEQ IDs NO: 14-135 in Table 1.AMINO ACID SEQUENCESTable 1

[0090] In some embodiments, the composition administered further comprises an adjuvant.

[0091] In some embodiments, the method further comprises administering an agent which modulates host immune tolerance. In some embodiment, the polypeptide administered is multimerized in a liposome or nanoparticle.

[0092] In some embodiments, the method further comprising administering one or more additional HIV-1 immunogens to induce a T cell response.

[0093] In some aspects, the invention provides a composition comprises a nanoparticle and a carrier, wherein the nanoparticle comprises an MPER peptide or HIV-1 envelope protein comprising a MPER peptide, wherein the MPER peptide or HIV-1 envelope protein comprising a MPER peptide is selected from SEQ ID NOS: 1-8, or 14-135 or any combination thereof. In some embodiments, the compositions comprise two, three, four or more different immunogens. In some embodiments the immunogens target different UCAs.

[0094] In some aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises a nucleic acid encoding one or more MPER peptides or HIV-1 envelope proteins comprising a MPER peptide from SEQ ID NOS: 1-8, or 14-135.

[0095] In some embodiments, the nanoparticle of the composition is a ferritin selfassembling nanoparticle.

[0096] In some aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the MPER peptides or HIV-1 envelope proteins comprising a MPER peptide, nucleic acids encoding an MPER peptide or HIV-1 envelope proteins comprising a MPER peptide, or compositions described herein. In some embodiments the methods comprise administering two, three, four or more different immunogens. In some embodiments, the different immunogens target different UCAs. In non-limiting embodiments the different immunogens are selected from SEQ ID NOS: 1-8, or 14-135.

[0097] In some embodiments, the composition is administered as a single prime or as repetitive immunization prime. In preferred embodiments, the repetitive immunization is administered 3 or 4 times.

[0098] In some embodiments, the composition is administered as a single boost or as a repetitive series of boosts. In preferred embodiments, the repetitive series of boosts is administered 3 or 4 times.

[0099] In some embodiments, the composition is a first composition administered as a prime. In some embodiments, the composition is a second composition administered as one or more boosts. In some embodiments, the method comprises administering the first composition as a prime and administering the second composition as one or more boosts. In preferred embodiments, the first composition and the second composition are different.

[0100] In some aspects, the invention provides a nucleic acid encoding any of the MPER peptides or HIV-1 envelope proteins comprising a MPER peptide described herein. In some embodiments, the invention provides a composition comprising the nucleic acid and a carrier.In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA is encapsulated in a lipid nanoparticle (LNP).

[0101] In some embodiments, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising the nucleic acid encoding any of the MPER peptides or HIV-1 envelope proteins comprising a MPER peptide described herein. In some embodiments, the immunogenic composition further comprises a carrier.

[0102] In certain aspects, the invention provides an immunogenic composition or composition, wherein the composition comprises at least two different HIV-1 MPER polypeptides or HIV-1 envelope proteins comprising a MPER peptide or nucleic acids encoding a HIV- 1 MPER polypeptide or HIV-1 envelope protein comprising a MPER peptide, or a combination thereof.

[0103] In certain aspects, the invention provides an immunogenic composition comprising a first immunogen and a second immunogen, wherein the first immunogen is a HIV-1 MPER peptide or HIV-1 envelope protein comprising a MPER peptide or a nucleic acid encoding said HIV-1 MPER peptide or HIV-1 envelope protein comprising a MPER peptide, and wherein the second immunogen is a different HIV-1 MPER peptide or HIV-1 envelope protein comprising a MPER peptide from SEQ ID NOS: 1-8, or 14-18 or 19-135 or a nucleic acid encoding said different HIV-1 MPER peptide or HIV-1 envelope protein comprising a MPER peptide In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering the immunogenic composition in an amount sufficient to induce an immune response. In certain embodiments, the method further comprising administering an agent which modulates host immune tolerance.COMPOSITIONS AND ANTIBODIES

[0104] In some aspects, this invention describes recombinant fusion protein sequences and immunogen displays (e.g., nanoparticles) displaying HIV fusion peptides or combinations thereof.

[0105] In one aspect this disclosure provides compositions including any one of the recombinant proteins, immunogen displays, nucleic acids, multimeric protein complexes, virus-like particles described herein, or a combination thereof, and a pharmaceutically acceptable carrier and an adjuvant.

[0106] The composition describe herein can be used for a prime boost immunization regimen. In certain embodiments the composition for a prime boost immunization regimen comprises one or more fusion peptides or portions thereof described herein.

[0107] In non-limiting embodiments, compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. In some embodiments, the compositions comprise a suitable carrier. Any suitable carrier can be use.

[0108] In certain aspects the compositions comprise a suitable adjuvant. Any suitable adjuvant can be used. In certain embodiments, TLR agonists are used as adjuvants. In certain embodiments, TLR agonists are TLR7 / 8 agonist. Non-limiting examples of TLR 7 / 8 are described in Evans et al. ACS Omega 2019. 4, 13, 15665-15677; Patinote et al. Eur J Med Chem. 2020 May 1; 193: 112238, Miller et al. in Front. Immunol., 10 March 2020 | https: / / doi.org / 10.3389 / fimmu.2020.00406. In certain embodiments, adjuvants are TLR 4 agonists. In some embodiments, the compositions include the TLR4 agonist GLA-SE as the adjuvant. In some embodiments, the composition is the TLR4 agonist GLA-SE as the adjuvant.

[0109] The compositions can include adjuvants, such as, for example but not limited to alum, 3M052, poly IC, MF-59 or another squalene-based oil-in-water emulsion adjuvant, AS01B, or other liposomal based adjuvant suitable for protein. In non-limiting embodiments, LNPs are used as adjuvants for immunogenic formulations comprising proteins, including multimeric protein complexes and nanoparticles. 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. In certain embodiments, AS04. a combination of alum and 3-0-desacyl-4'-monophosphoryl lipid A (MPL) developed by GSK. In certain embodiments the adjuvant is AS03, an oil-in water emulsion combination adjuvant developed by GSK. Non-limiting embodiments of adjuvants are described in the NIH 2018 Strategic Plan for Research on Vaccine Adjuvants, www.niaid.nih. gov / sites / default / files / NIAIDStrategicPlanVaccineAdjuvants2018. pdf, which is incorporated by reference herein for all purposes. 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.

[0110] The peptides, proteins, and / or nucleic acid immunogens of the invention can be formulated with, and / or administered with, adjuvants such as lipid A. oCpGs, TLR4 agonists or TLR 7 agonists that facilitate robust antibody responses (Rao et al, Immunobiol. Cell Biol.82(5):523 (2004)). Non-limiting examples of other adjuvants that can be used include alum and Q521 (which do not break existing B cell tolerance). In certain embodiments, formulations comprise an adjuvant that is designed to break forms of B cell tolerance, such as oCpGs in an oil emulsion such as Emulsigen (an oil in water emulsion) (Tran et al, Clin. Immunol. 109(3):278-287 (2003)).

[0111] Autoreactive B cells can be activated by TLR ligands through a mechanism dependent on dual engagement of the B cell receptor (BCR) and TLR (Leadbetter et al, Nature 416:603 (2002); Marshak-Rothstein et al, Annu. Rev. Immunol. 25: 419-41 (2007), Herlands et al, Immunity 29:249-260 (2008), Schlomchik, Immunity 28: 18-28 (2008)). In certain embodiments of immunogen design of the instant invention, soluble IFN-a is encapsulated into liposomes comprising MPER peptides such as MPER656 or MPER656- L669S peptides. IFN-a has been reported to modulate and relax the selectivity for autoreactive B cells by lowering the BCR activation threshold (Uccellini et al, J. Immunol. 181:5875-5884 (2008)). The design of the immunogens results from the observation that lipid reactivity of gp41MPER antibodies is required for both binding to membrane bound MPER epitopes and in the neutralization of HIV- 1.

[0112] The B cell subsets that the liposomes can target include any B cell subset capable of making polyreactive antibodies that react with the epitopes of the MPER. These B cell subsets include, but are not limited to, the marginal zone IgM+ CD27+ B cell subset (Weill et al, Annu. Rev. Immunol. 27:267-85 (2009), Li et al, J. Exp. Med 195: 181-188 (2002)), the transitional populations of human B cells (Sims et al, Blood 105:4390-4398 (2005)), and the human equivalent of the B cells that express the human equivalent of the mouse Immunoglobulin (Ig) light chain lambda X (Li et al, Proc. Natl. Acad. Sci. 103: 11264-11269 (2006). Witsch et al, J. Exp. Med. 203: 1761-1772 (2006)). All of these B cell subsets have the capacity to make multi-reactive antibodies and, therefore, to make antibodies that have the characteristic of reacting with both lipids and HIV-1 gp41. Other antibodies may be multi-reactive and react to different epitopes on the same MPER peptide, different epitopes on two different MPER peptides, an epitope of an MPER peptide and another epitope.

[0113] The peptide, proteins and / or nucleic acid immunogens can be administered, for example, IV, intranasally, subcutaneously, intraperitoneally, intravaginally, or intrarectally. The route of administration can vary', for example, with the patient, the conjugate and / or the effect sought, likewise the dosing regimen.METHODS FOR INDUCING AN IMMUNE RESPONSE

[0114] Aspects of the invention include methods for inducing an immune response to HIV-1 in a subject. The method can comprise administering to the subject an effective amount of the recombinant fusion protein, the immunogen display, the multimeric protein complex, the nucleic acids, the virus-like particles, or the immunogenic compositions describe herein, or combinations thereof, in an amount sufficient to induce an immune response in a subject.

[0115] The immune response against the compositions of the invention can be generated by one or more inoculations of a subject with an immunogenic composition of the invention. A first inoculation can be termed a “primary inoculation’" and subsequent immunizations can be termed “booster inoculations”. Booster inoculations can enhance the immune response, and immunization regimens including at least one booster inoculation can be used. Any composition of the invention may be used for a primary or booster immunization. The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, T cell populations can by monitored by conventional methods. The clinical condition of a subject can be monitored for the desired effect, e.g., limiting HIV-1 infection, improvement in disease state (e.g., reduction in viral load), etc. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the polypeptide, VLP, or composition, and / or adjuvant, can be increased or the route of administration can be changed.

[0116] In some embodiments, one or more of the following effects can result from the administration of “sufficient amount,” or “effective amount” of any of the immune inducing compositions described herein: (i) the reduction or amelioration of the severity of a viral infection and / or a symptom associated therewith; (ii) the reduction in the duration of a viral infection and / or a symptom associated therewith; (iii) the regression of a viral infection and / or a symptom associated therewith; (iv) the reduction of the titer of a virus; (v) the reduction in organ reduced function or failure associated with a viral infection; (vi) the reduction in hospitalization of a subject; (vii) the reduction in hospitalization length; (viii) the increase in the survival of a subject; (ix) the elimination of a virus infection; (x) the inhibition of the progression of a viral infection and / or a symptom associated therewith; (xi) the prevention of the spread of a virus from a cell, tissue or subject to another cell, tissue orsubject; and / or (xii) the enhancement or improvement the therapeutic effect of another therapy.

[0117] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the stabilized MPER peptides or HIV-1 envelope proteins comprising a MPER peptide of the invention. In certain embodiments, the composition is administered as a prime and / or a boost. In certain embodiments, the composition comprises nanoparticles. In certain embodiments, methods of the invention further comprise administering an adjuvant.

[0118] In certain aspects, the invention provides nucleic acids comprising sequences encoding polypeptides or proteins of the invention. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0119] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive MPER peptides or HIV-1 envelope proteins comprising a MPER peptide. 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.

[0120] In certain aspects the invention provides nucleic acids encoding the inventive polypeptide or protein designs. 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.

[0121] In non-limiting embodiments, the invention provides compositions comprising an MPER peptide or HIV-1 envelope proteins comprising a MPER peptide selected from SEQ ID NOS: 1-8, or 14-135, or any combination thereof. Provided are also methods of using these MPERs or HIV-1 envelope proteins comprising a MPER peptide and / or nucleic acids, and / or compositions comprising administering an amount sufficient to induce immune responses in a subject.

[0122] In some embodiments, the invention provides a nucleic acid or encoding a HIV-1 MPER polypeptide or HIV-1 envelope protein comprising a MPER peptide. In non-limiting embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA comprises the nucleic acids or any combination thereof., wherein thymine (T) will be uridine (U). In some embodiments, the mRNA comprises the nucleic acids, wherein thymine (T) will be 1- methyl-psuedouridine. In some embodiments, the mRNA is modified. In someembodiments, the modification is a modified nucleotide such as 5-methyl-cytidine and / or 6- methyl-adenosine and / or modified uridine. In some embodiments, the mRNA comprises a poly A tail about 85 to about 200 nucleotides long. In some embodiments, the mRNA comprises a poly A tail about 85 to about 110 nucleotides long. In some embodiments, the mRNA comprises a poly A tail about 90 to about 110 nucleotides long. In some embodiments, the mRNA comprises. In some embodiments, the mRNA comprises nucleic acids wherein thymine (T) will be I-methyl-psuedouridine and wherein the sequence comprises the nucleotides up to the poly A tail, wherein the mRNA comprises a poly A tail about 85 to about 200 nucleotides long. In some embodiments, the mRNA comprises the nucleic acids wherein thymine (T) will be 1-methyl-psuedouridine and wherein the sequence comprises the nucleotides up to the poly A tail, wherein the mRNA comprises a poly A tail about 85 to about 110 nucleotides long. In some embodiments, the mRNA comprises nucleic acids wherein thymine (T) will be 1-methyl-psuedouridine and wherein the sequence comprises the nucleotides up to the poly A tail, wherein the mRNA comprises a poly A tail about 90 to about 110 nucleotides long. In non-limiting embodiments, the mRNA is administered as an LNP.

[0123] In some embodiments, the invention provides an immunogenic composition comprising a nucleic acid encoding the HIV-1 MPER peptide or HIV-1 envelope protein comprising a MPER peptide and a carrier. In some embodiments, the compositions comprise at least two different immunogens targeting different UCAs. In non-limiting embodiments, the immunogens are from SEQ ID NOS: 1-8 and / or 14-135.

[0124] In some embodiments the immunogenic composition further comprises an adjuvant.

[0125] In some embodiments, the nucleic acid encoding one or more MPERs selected from SEQ ID NOS: 1-8 and 14-135, or any combination thereof is operably linked to a promoter. In some embodiments, the nucleic acid is inserted in an expression vector.

[0126] In some aspects, the invention provides a method of inducing an immune response in a subject comprising administering a composition comprising any suitable form of a nucleic acid(s) encoding one or more MPER peptide or HIV-1 envelope proteins comprising a MPER peptide selected from SEQ ID NOS: 1-8, or 14-135, or any combination thereof in an amount sufficient to induce an immune response.

[0127] In some embodiments, the composition administered comprises a nucleic acid encoding a soluble, stabilized, or a transmembrane bound MPER peptide.

[0128] In embodiments, a therapeutically effective amount can comprise less than about 0. 1 mg / kg, about 0. 1 mg / kg, about 0.5 mg / kg. about 1.0 mg / kg. about 2.5 mg / kg. about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg. about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg. about 300 mg / kg, about 325 mg / kg. about 350 mg / kg, about 375 mg / kg, about 400 mg / kg, about 425 mg / kg, about 450 mg / kg, about 475 mg / kg, about 500 mg / kg, about 525 mg / kg, about 550 mg / kg, about 575 mg / kg, about 600 mg / kg, about 625 mg / kg, about 650 mg / kg. about 675 mg / kg, about 700 mg / kg, about 725 mg / kg, about 750 mg / kg, about 775 mg / kg. about 800 mg / kg, about 825 mg / kg. about 850 mg / kg, about 875 mg / kg, about 900 mg / kg, about 1.0 g / kg, about 1.5 g / kg, about 2.0 g / kg, about 2.5 g / kg, about 5 g / kg, about 10 g / kg, about 25 g / kg, about 50 g / kg, or more than 50 g / kg of compound per body weight of a subject.

[0129] In embodiments, the therapeutically effective amount comprises less than about 0. 1 mg, about 0.1 mg, about 0.5 mg. about 1.0 mg, about 2.5 mg. about 5 mg, about 7.5 mg. about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 135 mg. about 150 mg, about 175 mg, about 200 mg, about 225 mg. about 250 mg, about 275 mg, about 300 mg, about 325 mg. about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g, or more than 50 g.

[0130] Aspects of the invention can comprise administering to a subject an immunogenic composition comprising an any of the HIV fusion peptides described herein.

[0131] In embodiments, administering can refer to providing a therapeutically effective amount of the composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fdlers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions,emulsions, or microemulsions: patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[0132] Embodiments can be administered to a subject in one or more doses. The dose level can vary' as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by a variety of means. For example, dosages can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration and can be decided according to the judgment of the practitioner and each patient's circumstances.

[0133] In some embodiments, multiple doses of the composition can be administered. The frequency of administration and the duration of administration of the composition can vary' depending on any of a variety7of factors, e.g., subject response, severity7of the symptoms, and the like. For example, in an embodiment, the pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every7other day (qod), daily (ad), twice a day (qid). three times a day (tid). or four times a day. In an embodiment, the pharmaceutical composition can be administered 1 to 4 times a day over a period of time, such as 1 to 10-day time period, or longer than a 10-day period of time.

[0134] In embodiments, the composition can be administered in combination with one or more additional active agents. For example, a first agent (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours. 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks. 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second agent (e.g., a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.NUCLEIC ACID SEQUENCES

[0135] In certain aspects the invention provides compositions and methods of MPER genetic immunization either alone or with MPER peptides or HIV-1 envelope proteins comprising a MPER peptide 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.

[0136] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See Graham BS, Enama ME, Nason MC, Gordon IJ, Peel SA, et al. (2013) DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for delivery of nucleic acids, as DNA and / or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments, DNA is formulated for delivery by a gene gun. In certain embodiments, DNA is administered byelectroporation, 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, 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 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.

[0137] 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 acidmolecules 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 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 technologies developed by In-Cell-Art.

[0138] In certain aspects, the invention provides nucleic acids comprising sequences encoding MPER peptides or HIV-1 envelope proteins comprising a MPER peptide 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.

[0139] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive MPER peptides or HIV-1 envelope proteins comprising a MPER peptide. 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.

[0140] Nucleic acid sequences provided herein are provided as DNA sequences.However, it should be understood that such sequences also represent RNA sequences, for example, mRNA. For example, RNA polymerase can be used to make RNA sequences from DNA sequences. In RNA sequences, thymine will be uridine. In some embodiments, uridine will be 1-methyl-pseudouridine. In some embodiments, nucleic acids of the invention, including RNA sequences or mRNAs, can further comprise any type of modified nucleotides, including, but not limited to 5-methyl-cytidine, 6-methyl-adenosine, or modified uridine.

[0141] Nucleic acid sequences provided herein, can be provided with a poly A tail length of 101 nucleotides. However, it should be understood that mRNA sequences can comprise different lengths of poly A tail. For example, in some embodiments the poly A tail is about 85 to about 200 nucleotides long. For example, in some embodiments the poly’ A tail is 85 to 200 nucleotides long. In some embodiments the poly A tail is about 85 to about 110 nucleotides long. In some embodiments the poly A tail is 85 to 110 nucleotides long. In some embodiments the poly A tail is about 90 to about 110 nucleotides long. In some embodiments the poly A tail is 90 to 1 10 nucleotides long.

[0142] In certain aspects the invention provides nucleic acids encoding the inventive MP ER peptides or HIV-1 envelope proteins comprising a MP ER peptide (e.g., any one of the MP ER peptides or MPER peptides or HIV-1 envelope proteins comprising a MP ER peptide disclosed in SEQ ID NOS: 1-8, or 14-135). In some embodiments, the one or more nucleic acids encoding any of the sequences of SEQ ID NOs: 1-8, or 14-135. 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.

[0143] In some embodiments the antibodies are administered as nucleic acids, including but not limited to mRNAs which could be modified and / or unmodified. See US Pub 20I80028645AI, 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.

[0144] 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.

[0145] 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. In some embodiments, the expression vector comprises DNA.

[0146] 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.

[0147] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the inventive sequences. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of the sequences of the invention, or a variant thereof or a fragment thereof. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of inventive antibodies. The RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.

[0148] In some embodiments, a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp. CleanCap® (e.g.. the AG, GG, AU, 3’OMe AG, or 3’OMe GG CleanCap®), or ARC A). 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.

[0149] 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).

[0150] Methods for in vitro transfection of mRNA and detection of MPER peptide expression are known in the art.

[0151] Methods for expression and immunogenicity determination of nucleic acid encoded MPER peptides are known in the art.

[0152] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. In certain embodiments recombinant proteins are produced in CHO cells.

[0153] The immunogenic MPER peptides or HIV-1 envelope proteins comprising a MPER peptide can also be administered as a protein boost in combination with a variety of nucleic acid MPER primes (e.g.. HIV-1 MPER peptides or HIV-1 envelope proteins comprising a MPER peptide delivered as DNA expressed in viral or bacterial vectors).

[0154] 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 hundreds of 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.

[0155] The compositions can be formulated in designs that incorporate appropriate carriers such as peptides for enhancing CD4+ T cell help, known as PADRE, GTH1, GTH2, or any combination thereof. In certain embodiments the compositions are delivered via intramuscular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes, or any other suitable route of immunization.HIV-1 ENVELOPE TRIMERS AND OTHER ENVELOPE DESIGNS

[0156] Elicitation of neutralizing antibodies is one goal for antibody-based vaccines. Neutralizing antibodies target the native trimeric HIV-1 Env on the surface virions. The trimeric HIV-1 envelope protein consists of three protomers each containing a gp!20 and gp41 heterodimer. Recent immunogen design efforts have generated soluble near-native mimics of the Env trimer that bind to neutralizing antibodies but not non-neutralizing antibodies. The recapitulation of the native trimer could be a key component of vaccine induction of neutralizing antibodies. Neutralizing Abs target the native trimeric HIV-1 Env on the surface of viruses (Poignard et al. J Virol. 2003 Jan;77(l):353-65; Parren et al. J Virol. 1998 Dec;72(12): 10270-4.; Yang et al. J Virol. 2006 Nov;80(22): l 1404-8.). The HIV-1 Env protein consists of three protomers of gpI20 and gp41 heterodimers that are noncovalently linked together (Center et al. J Virol. 2002 Aug;76(15):7863-7.). Soluble near-native trimers preferentially bind neutralizing antibodies as opposed to non-neutralizing antibodies (Sanders et al. PLoS Pathog. 2013 Sep; 9(9): e!003618).

[0157] The invention contemplates any other design, e.g. stabilized trimer, of the sequences described here in. For non-limiting embodiments of additional stabilized trimerssee US2015 / 0366961, US2020 / 0002383, US2021 / 0187091 and US2020 / 0113997, F14 and / or VT8 designs (US2021 / 0379177) all of which are incorporated by reference in their entirety.

[0158] Multimeric nanoparticles that comprise and / or display HIV envelope protein or fragments on their surface can be used a vaccine immunogens.

[0159] In some instances, the nucleic acid encoding an antigen (e.g., a HIV-1 envelope polypeptide comprising the MPER peptides described herein) is fused via a linker / spacer to a nucleic acids sequence encoding a protein which can self-assemble. Upon translation, a fusion protein is made that can self-assemble 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.

[0160] 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.

[0161] To improve the interaction between the naive B cell receptor and immunogens, in some embodiments, an 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.

[0162] Any suitable ferritin sequence could be used. In non-limiting embodiments, ferritin sequences are disclosed in US Patent 10,961,283, the content of which is hereby incorporated by reference in its entirety.

[0163] 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.

[0164] The nanoparticle immunogens are composed of various forms of HIV-1 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.

[0165] 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 tag or a N- terminal pentaglycine repeat tag is added to the envelope trimer gene. Cholesterol is also synthesized with these two tags. In a non-limiting embodiment, a C-terminal tag is LPXTGG, where X signifies any amino acid but most commonly Ala, Ser. Glu. 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.

[0166] The invention provides designs of envelopes and trimer designs wherein the envelope comprises a linker which permits addition of a lipid, such as but not limited to cholesterol, via a sortase A reaction. See e.g., Tsukiji, S. and Nagamune, T. (2009), Sortase- Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering. ChemBioChem, 10: 787-798. doi:10.1002 / cbic.200800724; Proft, T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation. 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):4411- 7; Pritz et al. J. Org. Chem. 2007, 72, 3909-3912.

[0167] The lipid modified envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0168] The lipid modified and multimerized envelopes and trimers could be formulated as liposomes. Any suitable liposome composition is contemplated.

[0169] 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.

[0170] 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, where X signifies any amino acid but most commonly Ala, Ser. Glu, or aN-terminal pentaglycine repeat tag is added to the envelope trimer gene. In a non-limiting embodiment, a C-terminal tag is LPXTGG, where X signifies any amino acid but most commonly Ala, Ser, Glu.

[0171] Provided are engineered MPER peptide immunogens derived from multiple viruses. Several strategies have been used to develop MPER peptides and encoding nucleic acids.

[0172] Modifications of nucleic acids encoding MPER peptides may include:• 5'UTR including aGcATAAAAGTCTCAACACAACATATACAAAACAAACGAATCTCAAGCAA TCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAA AAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCGCT (SEQ ID NO: xx). Without being bound by theory’, this modification is an improved 5' UTR sequence for mRNA stability and half-life from screens. See Messenger RNA-BasedVaccines Against Infectious Diseases. Alameh MG, Weissman D, Pardi N.Curr Top Microbiol Immunol. 2020 Apr 17. doi: 10.1007 / 82_2020_202. PMID: 32300916.• 3'UTR including actagtAGTGACTGACTAGGATCTGGTTACCACTAAACCAGCCTCAAGAACAC CCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTACAAAATGTT GTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTC TTCACATTCT (SEQ ID NO: xx). Without being bound by theory, this modification is an improved 5' UTR sequence for mRNA stability and half-life from screens. See Messenger RNA-Based Vaccines Against Infectious Diseases. Alameh MG, Weissman D. Pardi N.Curr Top Microbiol Immunol. 2020 Apr 17. doi: 10.1007 / 82_2020_202. PMID: 32300916.• poly A (immediately after 3'UTR) includesAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAA (SEQ ID NO: x). Without being bound by theory, this modification is an improved polyA tail sequence for mRNA stability and half-life. See Jalkanen et al. Semin Cell Dev Biol. 34:24-32 (2014).• mRNA codon optimization includes a reverse translation of protein amino acid sequence to optimal codons. Without being bound by theory, this modification codon optimization is performed as follow: amino acid sequence is reverse translated into an DNA sequence using a modified mammalian codon usage table. The table increases both the CIA and the GC content of the mRNA. The reverse translated sequence (or mRNA sequence) is modeled into mFold and Delta H / Delta G computed, and the sequence with the lowest free energy is selected. In some cases, the codons can be replaced in specific locations to relax the tridimentional structure of the optimized mRNA. The sequence is then cloned between the 5'UTR and 3'UTR above. See Leppek et al. Nature Communications 13: 1536 (2022).

[0173] The exemplary constructs provided herein, can include various combinations of these modifications. Any modification or combination of the modifications described herein, including but not limited, to different versions of soluble proteins, different versions of membrane expressed proteins, stabilization mutations, furin cleavage site mutations, signal peptides, and / or cytoplasmic tail modifications can be applied to any MPER peptide sequence or HIV-1 envelope protein sequence comprising a MPER peptide.

[0174] In certain aspects, the invention provides a nucleic acid comprising a sequence encoding the recombinant HIV-1 MPER peptide or HIV-1 envelope protein sequence comprising a MPER peptide described herein. In certain aspects, the invention provides an immunogenic composition comprising the MPER peptide or HIV-1 envelope protein sequence comprising a MPER peptide described herein and a carrier. In certain aspects, the invention provides an immunogenic composition comprising the nucleic acid described herein and a carrier. In some embodiments, the immunogenic composition described herein further comprises an adjuvant. In some embodiments, the nucleic acid is operably linked to a promoter, and optionally the nucleic acid is inserted in an expression vector.

[0175] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering a composition comprising any suitable form of a nucleic acid(s) described herein or the recombinant HIV-1 MPER peptide or HIV-1 envelope protein sequence comprising a MPER peptide described herein in an amount sufficient to induce an immune response. In some embodiments, the composition further comprises an adjuvant. In some embodiments, the method further comprises administering an agent which modulates host immune tolerance. In some embodiments, the nucleic acid administered is a mRNA. In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle. In some embodiments, the method further comprises administering one or more additional HIV-1 immunogens to induce a T cell response.

[0176] In certain aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the nucleic acids described herein. In some embodiments, the composition is an immunogenic composition. In some embodiments, the nucleic acid is a mRNA. In some embodiments, the nanoparticle is a lipid nanoparticle.

[0177] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising any one of the recombinant HIV-1 MPER peptides or HIV-1 envelope protein sequences comprising a MPER peptide or any one of the nucleic acids described herein or compositions described herein. In some embodiments, the composition is administered as a prime. In some embodiments, the composition is administered as a boost.

[0178] In certain aspects, the invention provides a composition comprising the nucleic acid described herein.

[0179] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering an immunogenic composition comprising therecombinant HIV-1 MPER peptide or HIV- 1 envelope protein sequence comprising a MPER peptide described herein or the nucleic acid described herein.

[0180] In some embodiments, the nucleic acid is a mRNA. In some embodiments, the mRNA is encapsulated in a lipid nanoparticle.

[0181] In certain aspects, the invention provides an immunogenic composition or composition described herein, wherein the composition comprises at least two different HIV- 1 MPER peptides or HIV-1 envelope sequences comprising a MPER peptide or nucleic acids encoding a HIV-1 MPER peptide, or HIV-1 envelope comprising a MPER peptide, or a combination thereof.

[0182] In some embodiments, the first immunogen and the second immunogen are both a HIV-1 MPER peptide or HIV-1 envelope protein sequence comprising a MPER peptide. In some embodiments, at least one of the first immunogen and the second immunogen is a nucleic acid. In some embodiments, the first immunogen and the second immunogen are both a nucleic acid.

[0183] In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA is encapsulated in an LNP.

[0184] In some embodiments, the immunogenic composition further comprises one or more additional immunogens, wherein the one or more additional immunogens is different to the first and second immunogens. In some embodiments, the immunogenic composition comprises a carrier. In some embodiments, the immunogenic composition further comprises an adjuvant.

[0185] In certain aspects, the invention provides a method of inducing an immune response in a subject comprising administering the immunogenic composition described herein in an amount sufficient to induce an immune response, in some embodiments, the method further comprises administering an agent which modulates host immune tolerance.

[0186] In certain aspects, the invention provides a composition comprising a nanoparticle and a carrier, wherein the nanoparticle comprises any one of the recombinant HIV-1 envelope described herein or the trimer described herein. In some embodiments, the composition is an immunogenic composition. In some embodiments, the nanoparticle is a ferritin selfassembling nanoparticle. In certain aspects, the invention provides a composition comprising the MPER peptide or HIV-1 envelope protein sequence comprising a MPER peptide described herein.EXAMPLES

[0187] 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.EXAMPLE 1: MPER-PEPTIDE LIPID IMMUNOGENS THAT CAN PRESENT BOTH PROXIMAL AND DISTAL MPER bnAb EPITOPES

[0188] A strategy was designed to create / identify prime and boost peptide-liposome immunogen designs that can bind to broad neutralizing antibodies (bnAbs) that recognized both proximal and distal MPER bnAb epitopes (FIG. 1).

[0189] In one embodiment, immunogens were designed with the following criteria: a) include lipids in both prime and boost immunogens; b) present both proximal and distal gp41 MPER bnAb epitopes; c) bind with an affinity gradient to each of the intermediate antibody in the bnAb maturation pathway; and d) target certain functional and improbable mutations in key intermediates.

[0190] MPER656 peptide-liposome (HVTN133 clinical trial immunogen) variants that bind to the distal MPER bnAb DH511.

[0191] Liposomes carrying the MPER656 peptide (FIG. 2A) can bind the bnAbs 4E10 and 10E8 (PMID: 22140469; 19640992), both broad neutralizing antibodies that recognize the distal conserved region of MPER. However, MPER656 peptide-liposomes cannot bind to lineage members of the DH511 antibody, an antibody that also binds to distal epitopes, because the MPER656 peptide does not include residues such as D674 (FIG. 1A) that make up the DH511 core epitope. Therefore, the distal MPER656 epitope was optimized for DH511 binding by introducing into its sequence these residues.

[0192] Four MPER656 peptide-liposomes carrying different variants of the MPER656 peptide (MPER656, MPER656.1, MPER656.2 and MPER656.3) were designed and assessed for binding to bnAbs known to bind to the distal (DH511.2) and proximal (2F5) regions of MPER.

[0193] As shown in FIG. 2A (table), two variants, MPER656. 1 and MPER656.3, bound to distal DH511 bnAb. In addition, as shown in FIG. 2B, among MPER656. 1 and MPER656.3 variants, MPER656. 1 showed the strongest binding to distal DH511.2 bnAb (a member of the DH511 lineage of antibodies). No binding of MPER656 liposomes to DH511.2 bnAb wasobserved. FIG. 2C shows that proximal 2F5 bnAb bound to each of the MPER656 liposomes and that binding of 2F5 to MPER656. 1 liposomes was retained and comparable to the binding of 2F5 to MPER656 liposomes.

[0194] A next generation design (Variant 4) of HVTN133 MPER liposomes immunogen binds with increasing strength to both the proximal and distal gp41 MPER bnAbs.EXAMPLE 2: DH1317 LINEAGE mAbs BINDING TO MPER656 VARIANT LIPOSOMES

[0195] Variants of the MPER656.1 peptide sequences were designed to optimize MPER bnAb epitopes based on the neutralization breadth signatures of the proximal (DFI1317.4) and the distal (DH51 1) MPER bnAbs. The MPER 656.1 variant peptide constructs shown in FIG.3 was used to develop four variants of the MPER656.1 peptide-liposome: UCA.OPT (also called variant 1), UCA.OPT2 (also called variant 2), DUAL. UCA.OPT (also called variant 3) and DUAL.UCA.OPT2 (also called variant 4). Binding of DH1317 lineage mAbs to all four MPER656. 1 variant liposomes was assessed.

[0196] FIG. 4A and 4B show that among the four MPER peptide-liposomes, and when compared to the MPER656 liposomes, variant 4 gave the highest level of binding to each of the DH1317 mAbs, including DH1317 UCA and the IA4 intermediate. In addition. FIG. 5A and 5B showed enhanced binding of DH1317 UCA or the IA4 intermediate to Variant 4 peptide-liposomes when compared to MPER656.1 liposomes. DH1317 IA4, the UCA- proximal intermediate, includes two functional and improbable mutations and is a key intermediate for vaccine target.

[0197] Binding to DH1317 intermediate (IA4) with two key functional and improbable mutations was markedly increased when compared to the MPER 656.1 liposomes.EXAMPLE 3: DH511 LINEAGE AB BINDING TO MPER656.1 VARIANT LIPOSOMES

[0198] Binding of different DH511 lineage mAbs to all four MPER656.1 variant liposomes was also assessed. As shown in FIG. 6A and 6B, among the four MPER656. 1 peptide-liposome variants, and when compared to the MPER656. 1 peptide-liposomes, variant4 showed the highest levels of binding to the affinity -matured DH511 IAS and to the bnAb DH511.2.

[0199] The variant 4 MPER liposomes showed enhanced binding to IAs of the DH511 bnAb lineage.EXAMPLE 4: ANTIBODY BINDING TO MRNA ENCODED CELLSURFACE EXPRESSED MPER

[0200] An mRNA construct with MPER + transmembrane domain (SEQ ID NO: 13) was used to transiently transfect 293 cells. The cell surface expressed MPER was detected bystaining with a panel of antibodies indicated in FIG. 7.

[0201] An mRNA-encoded MPER construct gave strong cell surface binding to both proximal and distal bnAbs.EXAMPLE 5: SUMMARY

[0202] A next generation design (Variant 4) of HVTN133 MPER liposomes immunogen binds with increasing strength to both the proximal and distal gp41 MPER bnAbs.

[0203] Binding to DH1317 intermediate (IA4) with two key functional and improbable mutations was markedly increased when compared to the MPER 656.1 liposomes.

[0204] The variant 4 MPER liposomes showed enhanced binding to lAs of the DH511 bnAb lineage.

[0205] An mRNA-encoded MPER construct gave strong cell surface binding to both proximal and distal bnAbs.EQUIVALENTS

[0206] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMSWhat is claimed:

1. A membrane proximal external region (MPER) immunogen from HIV to which: a) an antibody from the DH511 lineage binds; or b) an antibody from the DH511 and an antibody from the 2F5 lineage binds; or c) an antibody from a DH1317 lineage and an antibody from a DH511 lineage binds.

2. An MPER immunogen from HIV, comprising an amino sequence:NEQDLLALDKWASLWNWFDISNWLWYIK (MPER656.E SEQ ID NO: 2);NEKDLLALDSWKNLWNWFSITKWLWYIK (MPER656.2; SEQ ID NO: 3); NEQELLALDKWNNLWS WFDITNWLWYIR (MPER656.3 ; SEQ ID NO: 4); NEQDLLELDKWASLWNWFDITKWLWYIK (UCA.OPT or Variant 1; SEQ ID NO: 5);NEQDLLALDKWASLWNWFDITNWLWYIK (UCA.OPT or Variant 2; SEQ ID NO: 6);NEQELLELDKWASLWNWFDITKWLWYIK (DUAL.UCA.OPT or Variant 3;SEQ ID NO: 7;NEQELLELDKWASLWNWFDITNWLWYIK (DUAL.UCA.OPT2 or Variant 4; SEQ ID NO: 8);NEQELLELDKWASLHNWTSITNWLWYIK (Ml; SEQ ID NO: 14);NEQELLELDKWASLWNWFDISNWGHYFA (M2; SEQ ID NO: 15);NEQELLELDKWASLHNWTSISNWGHYFA (M3; SEQ ID NO: 16);NEQELLELDKWASLWNWFDITNWIWYRWIFRG (M4; SEQ ID NO: 17); and NEQELLWLDKWDSLHPWTSITPWLWYIK (M5; SEQ ID NO: 18); and / or an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to any one of SEQ ID NOs: 1-8, and 14-18.

3. An MPER immunogen from HIV, comprising an amino acid sequence selected from the group consisting of of SEQ ID NO: 19-135, or an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 19-135.

4. A lipid immunogen display comprising one or more of the MPER immunogens fromany one of claims 1-3.

5. The lipid immunogen display of claim 4, comprising a liposome.

6. A nucleic acid encoding the MP ER immunogen from any one of claims 1-3.

7. A cell comprising a nucleic acid encoding the MPER immunogen of any one of claims 1-3.

8. The cell of claim 7, displaying the MPER immunogen on the cell surface.

9. A lipid immunogen display that displays an MPER immunogen from HIV on an exterior surface, the MPER immunogen comprising an amino sequence:NEQDLLALDKWASLWNWFDISNWLWYIK (MPER656.1; SEQ ID NO: 2);NEKDLLALDSWKNLWNWFSITKWLWYIK (MPER656.2; SEQ ID NO: 3); NEQELLALDKWNNLWSWFDITNWLWYIR MPER656.3; SEQ ID NO: 4); NEQDLLELDKWASLWNWFDITKWLWYIK (UCA.OPT or Variant 1; SEQ ID NO: 5);NEQDLLALDKWASLWNWFDITNWLWYIK (UCA.OPT or Variant 2; SEQ ID NO: 6);NEQELLELDKWASLWNWFDITKWLWYIK (DUAL.UCA.OPT or Variant 3;SEQ ID NO: 7;NEQELLELDKWASLWNWFDITNWLWYIK (DUAL.UCA.OPT2 or Variant 4; SEQ ID NO: 8);NEQELLELDKWASLHNWTSITNWLWYIK (Ml; SEQ ID NO: 14);NEQELLELDKWASLWNWFDISNWGHYFA (M2; SEQ ID NO: 15);NEQELLELDKWASLHNWTSISNWGHYFA (M3; SEQ ID NO: 16);NEQELLELDKWASLWNWFDITNWIWYRWIFRG (M4; SEQ ID NO: 17); and NEQELLWLDKWDSLHPWTSITPWLWYIK (M5; SEQ ID NO: 18); or an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to any one of SEQ ID NOs: 1-8, and 14-18.

10. A lipid immunogen display that displays an MPER immunogen from HIV on an exterior surface, the MPER immunogen selected from the group consisting of SEQ ID NO: 19-135, oran amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 19-135.

11. A method for stimulating anti -HIV (anti -HIV- 1) antibodies in a subject using a primeboost immunization, comprising: priming with an immunogen to produce unmutated common ancestor antibodies (UCAs); and boosting with one or more immunogens to produce early intermediate antibodies (IA), late IAS and broadly neutralizing antibodies (bnAbs).

12. The method of claim 11. wherein; a priming immunogen comprises an amino acid sequence of one or more of SEQ ID NO: 1-8 or SEQ ID NO: 1-8 or 14-135; and a boosting immunogen comprises an amino acid sequence of one or more of SEQ ID NO: 1-8 and / or an mRNA-encoded MPER.

13. The method of claim 11 or 12 wherein: a priming immunogen comprises MPER656.1 Variant 1; and a boosting immunogen comprises MPER656.1 V ariant 2, MPER656. 1 or an mRNA-encoded MPER / transmembrane domain (TMD).

14. The method of claim 11 or 12, wherein: a priming immunogen comprises MPER656. 1 ; and a boosting immunogen comprises MPER 56. 1 Variant 4 or an mRNA-encoded MPER / TMD.

15. A broadly neutralizing antibody stimulated by the MPER immunogens of any one of claims 1-3, the lipid immunogen display of any one of claims 4-5 and 9-10, the nucleic acid of claim 6. the cell of claims 7 or 8, or the methods of claims 11-14.

16. An immunogen display, comprising an amino sequence:NEQDLLALDKWASLWNWFDISNWLWYIK (MPER656.1: SEQ ID NO: 2); NEKDLLALDSWKNLWNWFSITKWLWYIK (MPER656.2; SEQ ID NO: 3); NEOELLALDKWNNLWS WFDITNWLWYIR (MPER656.3 ; SEQ ID NO: 4);NEQDLLELDKWASLWNWFDITKWLWYIK (UCA.OPT or Variant 1; SEQ IDNO: 5);NEQDLLALDKWASLWNWFDITNWLWYIK (UCA.OPT or Variant 2; SEQ ID NO: 6);NEQELLELDKWASLWNWFDITKWLWYIK (DUAL.UCA.OPT or Variant 3;SEQ ID NO: 7;NEQELLELDKWASLWNWFDITNWLWYIK (DUAL.UCA.OPT2 or Variant 4; SEQ ID NO: 8);NEQELLELDKWASLHNWTSITNWLWYIK (Ml; SEQ ID NO: 14);NEQELLELDKWASLWNWFDISNWGHYFA (M2; SEQ ID NO: 15);NEQELLELDKWASLHNWTSISNWGHYFA (M3; SEQ ID NO: 16);NEQELLELDKWASLWNWFDITNWIWYRWIFRG (M4; SEQ ID NO: 17); and NEQELLWLDKWDSLHPWTSITPWLWYIK (M5; SEQ ID NO: 18); or an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to any one of SEQ ID NOs: 1-8, and 14-18.

17. An immunogen display, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19-135, or an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 19-135.

18. The immunogen display of one of claims 16 or 17, comprising a protein scaffold, nanoparticle or virus-like particle.

19. The immunogen display of one of claims 16 or 17 comprising a self-assembling protein nanoparticle.

20. The immunogen display of one of claims 16 or 17, that does not include a lipid.

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