HIV immunogenic polypeptides and vaccines and uses thereof

Abstract

The present disclosure relates to HIV immunogenic polypeptides, polynucleotides encoding such HIV immunogenic polypeptides, self-amplifying messenger RNA (SAM) vectors comprising such polynucleotides, and viral vectors comprising such polynucleotides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 388,599, filed on July 12, 2022, U.S. Provisional Application No. 63 / 384,020, filed on November 16, 2022, and U.S. Provisional Application No. 63 / 485,122, filed on February 15, 2023, each of which is incorporated herein in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted in .XML file format and is hereby incorporated by reference in its entirety. The .XML copy was created on June 27, 2023, is named 1444-WO-PCT.xml, and is 691,866 bytes in size. Technical Field

[0005] The present disclosure relates to novel HIV immunogens, polynucleotides encoding such HIV immunogens, and HIV vaccines comprising adenoviral vectors and / or self-amplifying messenger RNA (SAM) vectors. These HIV vaccines may comprise chimpanzee adenovirus (ChAd) vectors and / or SAM vectors. Background Art

[0006] Human immunodeficiency virus type 1 (HIV-1) infection causes global morbidity and mortality, affecting 38 million people worldwide and 1.1 million in the United States (US) (Joint United Nations Programme on HIV / AIDS (UNAIDS), UNAIDS Data, Geneva, Switzerland, 2020). Antiretroviral therapy (ART) is highly effective in suppressing viral replication in people living with HIV (PWH). However, discontinuation of ART leads to a rapid rebound of viral replication; therefore, PWH must continue ART throughout their lives. Rebound viremia arises from persistent reservoirs of HIV-infected cells containing replication-competent, full-length proviral HIV DNA that cannot be eliminated by ART. The HIV reservoir is established very early in HIV infection, and a large portion of this reservoir is transcriptionally silent (i.e., latent) at any given time. Consequently, the latent HIV reservoir cannot be recognized and eliminated by the immune system. Latent HIV provirus can be found in all CD4+ T cell subsets (including long-lived memory cells), monocytes, and macrophages in peripheral blood and tissues. Viral rebound is likely due to the stochastic reactivation of latently infected cells in the absence of viral suppression (Sengupta and Siliciano, Targeting the Latent Reservoir for HIV-1. Immunity 2018; 48(5): 872-95). Therefore, there is a need to develop new therapies that can target and destroy latently infected HIV cells (a major obstacle to HIV cure) to reduce and ultimately eliminate HIV reservoirs, thereby achieving long-term viral remission. Summary of the Invention

[0007] Disclosed herein are polynucleotides comprising any one of the HIV immunogen nucleic acid sequences SEQ ID NOs: 524-526.

[0008] Disclosed herein are polynucleotides comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the HIV immunogen nucleic acid sequences SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequences SEQ ID NOs: 524-526.

[0009] Disclosed herein are polynucleotides comprising an HIV immunogen nucleic acid sequence encoding any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

[0010] Disclosed herein are polynucleotides comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the nucleic acid sequence encoding the polypeptide of any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

[0011] In some embodiments, the polynucleotide comprises cDNA or mRNA.

[0012] Disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising any of the polynucleotides disclosed herein.

[0013] In some embodiments, the SAM comprises a polynucleotide comprising the HIV immunogen nucleic acid sequence of any one of SEQ ID NOs: 524-526.

[0014] In some embodiments, the SAM comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the HIV immunogen nucleic acid sequences SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequences SEQ ID NOs: 524-526.

[0015] In some embodiments, the SAM comprises a polynucleotide comprising an HIV immunogen nucleic acid sequence encoding any one of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528.

[0016] In some embodiments, the SAM comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528, wherein the percent identity spans the entire length of the nucleic acid sequence encoding a polypeptide of any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

[0017] In some embodiments, the SAM is derived from a virus.

[0018] In some embodiments, the virus is an RNA virus.

[0019] In some embodiments, the RNA virus is a positive-sense single-stranded RNA virus.

[0020] In some embodiments, the virus is selected from the group consisting of an alphavirus, a flavivirus, a nidovirus, a nodavirus, and a picornavirus.

[0021] In some embodiments, the alphavirus is selected from the group consisting of an Old World (OW) alphavirus and a New World (NW) alphavirus.

[0022] In some embodiments, the OW alphavirus is selected from Chikungunya virus (CHIKV), Ross River virus (RRV), Semliki Forest virus (SFV), and Sindbis virus (SINV).

[0023] In some embodiments, the NW alphavirus is selected from Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and Western equine encephalitis virus (WEEV).

[0024] In some embodiments, the SAM is derived from Venezuelan equine encephalitis virus (VEEV).

[0025] In some embodiments, the virus comprises a modified viral genome.

[0026] In some embodiments, the modified viral genome comprises a deletion of one or more genes encoding one or more viral structural proteins.

[0027] In some embodiments, a modified viral genome is generated by replacing one or more viral structural proteins with any of the polynucleotides disclosed herein.

[0028] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising the nucleic acid sequences of SEQ ID NOs: 524-526.

[0029] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence of SEQ ID NOs: 524-526.

[0030] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the polypeptides of SEQ ID NOs: 524-526.

[0031] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0032] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0033] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0034] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of the polypeptide of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0035] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide encoding the polypeptide of any one of SEQ ID NOs: 1-344.

[0036] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 1-344.

[0037] In some embodiments, the modified viral genome is generated by replacing one or more viral structural proteins with a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of the polypeptides of SEQ ID NOs: 1-344.

[0038] In some embodiments, the virus is replication deficient.

[0039] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide comprises any one of the nucleic acid sequences of SEQ ID NOs: 522-523.

[0040] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence SEQ ID NOs: 522-523.

[0041] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide comprises any one of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0042] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence SEQ ID NOs: 524-526.

[0043] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide encodes any one of the amino acid sequences of SEQ ID NOs: 527-528.

[0044] Also disclosed herein are self-amplifying RNA (SAM or saRNA) comprising the following polynucleotide, wherein the polynucleotide encodes a polypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528.

[0045] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0046] Also disclosed herein is a self-amplifying RNA (SAM or saRNA) comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity is across the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0047] In some embodiments, the third polynucleotide sequence encodes two or more polypeptide fragments.

[0048] In some embodiments, the third polynucleotide sequence encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NOs: 1-344.

[0049] In some embodiments, one or more of these polypeptide fragments are contiguous or fused to an adjacent polypeptide fragment.

[0050] In some embodiments, one or more of the polypeptide fragments are joined to an adjacent polypeptide fragment via one or more peptide linkers.

[0051] In some embodiments, the one or more peptide linkers are selected from one or more of the following: a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linking sequence, and combinations thereof.

[0052] In some embodiments, the polyalanine linker comprises or consists of 2 or 3 consecutive alanine residues, e.g., AA, AAA, AAY, or AAX, where X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y).

[0053] In some embodiments, the flexible linker or polyglycine linker comprises or consists of GG, GGS, GSG, or GGGS (SEQ ID NO: 421).

[0054] In some embodiments, the cleavable linker is selected from a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and trichothivirus (T2A)), a furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), a Nef linker sequence, and combinations, derivatives, or variants thereof.

[0055] In some embodiments, the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382), and RRKR (SEQ ID NO: 383).

[0056] In some embodiments, the cleavable linker comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388).

[0057] In some embodiments, the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388).

[0058] In some embodiments, the Nef linker sequence comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), or GALDI (SEQ ID NO: 391), wherein the percent identity spans the entire length of SEQ ID NOs: 389-391.

[0059] In some embodiments, the Nef linker sequence comprises or consists of an amino acid sequence selected from the group consisting of VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), and GALDI (SEQ ID NO: 391).

[0060] In some embodiments, the promoter sequence comprises the polynucleotide sequence of SEQ ID NO:529.

[0061] Also disclosed herein are expression cassettes comprising any of the polypeptides disclosed herein or a SAM operably linked to one or more regulatory sequences.

[0062] In some embodiments, the expression cassette comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NOs: 524-526 operably linked to one or more regulatory sequences.

[0063] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of SEQ ID NOs: 524-526 operably linked to one or more regulatory sequences.

[0064] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding a polypeptide of any one of SEQ ID NOs: 524-526, wherein the percent identity spans the entire length of the polypeptide of SEQ ID NOs: 524-526, and wherein the polynucleotide is operably linked to one or more regulatory sequences.

[0065] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, and is operably linked to one or more regulatory sequences.

[0066] In some embodiments, the expression cassette comprises a polynucleotide encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, and is operably linked to one or more regulatory sequences.

[0067] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, and is operably linked to one or more regulatory sequences.

[0068] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding a polypeptide of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity spans the entire length of the polypeptide of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the polynucleotide is operably linked to one or more regulatory sequences.

[0069] In some embodiments, the expression cassette comprises a polynucleotide encoding the polypeptide of any one of SEQ ID NOs: 1-344 and is operably linked to one or more regulatory sequences.

[0070] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 1-344 and operably linked to one or more regulatory sequences.

[0071] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence encoding a polypeptide of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of the polypeptide of SEQ ID NOs: 1-344, wherein the polynucleotide is operably linked to one or more regulatory sequences.

[0072] In some embodiments, the polynucleotide is operably linked to and under the control of a constitutive promoter.

[0073] In some embodiments, the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, an EF1a promoter, and a 26S subgenomic promoter.

[0074] In some embodiments, the promoter comprises any one of the promoter sequences SEQ ID NOs: 529-530.

[0075] Also disclosed herein are lipid nanoparticles (LNPs) comprising any of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0076] In some embodiments, the LNP comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0077] In some embodiments, the LNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0078] In some embodiments, the LNP comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0079] In some embodiments, the LNP comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0080] In some embodiments, the LNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0081] In some embodiments, the LNP comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0082] In some embodiments, the LNP comprises a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0083] In some embodiments, the LNP comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0084] In some embodiments, the LNP comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0085] In some embodiments, the LNP comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0086] In some embodiments, the LNP comprises or is synthesized from an ionizable lipid, a phospholipid, cholesterol, a PEGylated lipid, or any combination thereof.

[0087] In some embodiments, the LNP comprises, or is synthesized from, the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0088] Also disclosed herein are polymeric nanoparticles (PNPs) comprising any of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0089] In some embodiments, the PNP comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0090] In some embodiments, the PNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0091] In some embodiments, the PNP comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0092] In some embodiments, the PNP comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0093] In some embodiments, the PNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0094] In some embodiments, the PNP comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0095] In some embodiments, the PNP comprises a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0096] In some embodiments, the PNP comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity spans the entire length of the amino acid sequence of SEQ ID NOs: 527-528, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0097] In some embodiments, the PNP comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs. NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0098] In some embodiments, the PNP comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0099] In some embodiments, the PNPs comprise or are synthesized from a non-degradable polymer, a degradable polymer, a natural material, a synthetic material, a monomer, a dendrimer, or any combination thereof.

[0100] In some embodiments, the PNPs comprise or are synthesized from poly(ethylene glycol) (PEG), poly(dimethylsiloxane) (PDMS), polyethyleneimine (PEI), poly(amidoamine) (PAMAM), poly(dimethylaminoethyl)acrylate (pDMAEA), orthenine-derived dendrimers, or any combination thereof.

[0101] In some embodiments, the PNP comprises, or is synthesized from, a mannosylated-PEI polyplex.

[0102] In some embodiments, the PNP comprises, or is synthesized from, a bioreducible linear cationic polymer (pABOL).

[0103] Also disclosed herein are nanoemulsions comprising any of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0104] In some embodiments, the nanoemulsion comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0105] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0106] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector containing such a polynucleotide.

[0107] In some embodiments, the nanoemulsion comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0108] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0109] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector containing such a polynucleotide.

[0110] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0111] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector containing such a polynucleotide.

[0112] In some embodiments, the nanoemulsion comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0113] In some embodiments, the nanoemulsion comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequence that is any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0114] In some embodiments, the nanoemulsion is a water-in-oil emulsion.

[0115] In some embodiments, the nanoemulsion comprises squalene, sorbitan trioleate, polysorbate 80, DOTAP, or any combination thereof.

[0116] Also disclosed herein are expression vectors comprising any of the polynucleotides, SAMs, or expression cassettes disclosed herein.

[0117] In some embodiments, the expression vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM or expression cassette comprising such a polynucleotide.

[0118] In some embodiments, the expression vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM or expression cassette comprising such a polynucleotide.

[0119] In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523, or a SAM or expression vector containing such a polynucleotide.

[0120] In some embodiments, the expression vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM or expression cassette comprising such a polynucleotide.

[0121] In some embodiments, the expression vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM or expression cassette comprising such a polynucleotide.

[0122] In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526, or a SAM or expression vector containing such a polynucleotide.

[0123] In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 527-528, or a SAM or expression cassette comprising such a polynucleotide.

[0124] In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528, or a SAM or expression cassette containing such a polynucleotide.

[0125] In some embodiments, the expression vector comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0126] In some embodiments, the expression vector comprises the following SAM, which comprises: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0127] In some embodiments, the expression vector is a plasmid vector, a bacterial vector, or a viral expression vector.

[0128] In some embodiments, the expression vector is a viral expression vector.

[0129] In some embodiments, the viral expression vector is a DNA virus or an RNA virus.

[0130] In some embodiments, the viral expression vector is replication-defective, replication-deficient, replication-attenuated, or replication-competent.

[0131] In some embodiments, the viral expression vector is from a virus selected from the group consisting of an adenovirus, an adeno-associated virus, an arenavirus, an alphavirus, a poxvirus, a cytomegalovirus, a rhabdovirus, a vesicular stomatitis virus, a flavivirus, a Maraba virus, and a vaccinia virus.

[0132] In some embodiments, the viral expression vector is from a virus from a taxonomic family selected from the group consisting of Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia viruses, e.g., Modified Vaccinia Virus Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculoviruses, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphaviruses, e.g., Venezuelan equine encephalitis virus).

[0133] In some embodiments, the viral expression vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Juninvirus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

[0134] In some embodiments, the viral expression vector is an arenavirus vector selected from: lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus).

[0135] In some embodiments, the arenavirus vector comprises a bi-segmented genome.

[0136] In some embodiments, the arenavirus vector comprises a three-segmented genome.

[0137] In some embodiments, the viral expression vector is a human adenovirus or a simian adenovirus.

[0138] In some embodiments, the simian adenovirus is selected from the group consisting of a chimpanzee adenovirus, a gorilla adenovirus, and a rhesus adenovirus.

[0139] In some embodiments, the viral expression vector is an adenovirus vector selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (ChAd), gorilla adenovirus, and rhesus monkey adenovirus.

[0140] In some embodiments, a ChAd is selected from the group consisting of ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22), ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (AdC28), ChAdY27 (AdC28), ChAdY30 (AdC31), ChAdY31 (AdC32), ChAdY32 (AdC33), ChAdY33 (AdC34), ChAdY34 (AdC35), ChAdY35 (AdC36), ChAdY36 (AdC37), ChAdY37 (AdC38), ChAdY38 (AdC39), ChAdY40 (AdC41), ChAdY41 (AdC42), ChAdY42 (AdC43), ChAdY43 (AdC44), ChAdY44 (AdC45), ChAdY45 (AdC46), ChAdY46 (AdC47), ChAdY47 (AdC48), ChAdY48 (AdC49), ChAdY50 (AdC49), ChAdY51 (AdC41), ChAdY52 (AdC43), ChAdY53 (AdC47), ChAdY54 (AdC49), ChAdY61 (AdC49), ChAdY62 (AdC41), ChAdY63 (AdC43), ChAdY71 (AdC49), ChAdY72 (AdC41), ChAdY73 (AdC43), ChAdY74 (AdC45), ChAd Ad30 (AdC30), ChAd31 (AdC31), ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 (AdC55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAd143 (AdC143), ChAd144 (AdC144), ChAd145 (AdC145), and ChAd147 (AdC147).

[0141] In some embodiments, the ChAd is ChAd68.

[0142] In some embodiments, the gorilla adenovirus is selected from GC44, GC45, and GC46.

[0143] In some embodiments, the rhesus adenovirus (RhAd) is selected from RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, and RhAd66.

[0144] In some embodiments, the human adenovirus or simian adenovirus comprises modifications of one or more adenoviral genes.

[0145] In some embodiments, the one or more adenoviral genes are selected from E1, E3, and E4 ORF2-4.

[0146] In some embodiments, the modification comprises deleting or substituting one or more nucleotides in one or more adenoviral genes, or inserting one or more nucleotides into one or more adenoviral genes.

[0147] In some embodiments, the human adenovirus or simian adenovirus comprises a deletion of at least a portion of the El region.

[0148] In some embodiments, the polynucleotide is inserted into a deletion of at least a portion of the El region.

[0149] In some embodiments, the expression vector comprises any one of the nucleic acid sequences SEQ ID NOs: 520-521.

[0150] In some embodiments, the expression vector comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 520-521, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 520-521.

[0151] Also disclosed herein is an expression vector comprising any one of the nucleic acid sequences SEQ ID NOs: 524-526.

[0152] Also disclosed herein is an expression vector comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0153] Also disclosed herein is an expression vector comprising a polynucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 527-528.

[0154] Also disclosed herein is an expression vector comprising a polynucleotide sequence encoding a polynucleotide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528.

[0155] Also disclosed herein is an expression vector comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528; (d) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (e) a polyA sequence; and (f) a fifth polynucleotide sequence comprising nucleotides 4620 to 32350 of SEQ ID NO: 520.

[0156] Also disclosed herein is an expression vector comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (f) a polyA sequence; and (g) a fifth polynucleotide sequence comprising nucleotides 4620 to 32350 of SEQ ID NO: 520.

[0157] In some embodiments, the third polynucleotide sequence encodes two or more polypeptide fragments.

[0158] In some embodiments, the third polynucleotide sequence encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NOs: 1-344.

[0159] In some embodiments, one or more of these polypeptide fragments are contiguous or fused to an adjacent polypeptide fragment.

[0160] In some embodiments, one or more of the polypeptide fragments are joined to an adjacent polypeptide fragment via one or more peptide linkers.

[0161] In some embodiments, the one or more peptide linkers are selected from one or more of the following: a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linking sequence, and combinations thereof.

[0162] In some embodiments, the polyalanine linker comprises or consists of 2 or 3 consecutive alanine residues, e.g., AA, AAA, AAY, or AAX, where X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y).

[0163] In some embodiments, the flexible linker or polyglycine linker comprises or consists of GG, GGS, GSG, or GGGS (SEQ ID NO: 421).

[0164] In some embodiments, the cleavable linker is selected from a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and trichothivirus (T2A)), a furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), a Nef linker sequence, and combinations, derivatives, or variants thereof.

[0165] In some embodiments, the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382), and RRKR (SEQ ID NO: 383).

[0166] In some embodiments, the cleavable linker comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388), wherein the percent identity spans the entire length of SEQ ID NOs: 384-388.

[0167] In some embodiments, the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388).

[0168] In some embodiments, the Nef linker sequence comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), or GALDI (SEQ ID NO: 391), wherein the percent identity spans the entire length of SEQ ID NOs: 389-391.

[0169] In some embodiments, the Nef linker sequence comprises or consists of an amino acid sequence selected from the group consisting of VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), and GALDI (SEQ ID NO: 391).

[0170] In some embodiments, the promoter sequence comprises the polynucleotide sequence of SEQ ID NO:529.

[0171] Also disclosed herein are viral vectors comprising any of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0172] In some embodiments, the viral vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0173] In some embodiments, the viral vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0174] In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0175] In some embodiments, the viral vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0176] In some embodiments, the viral vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0177] In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0178] In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0179] In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the amino acid sequence of SEQ ID NOs: 527-528, or a SAM, expression cassette or expression vector containing such a polynucleotide.

[0180] In some embodiments, the viral vector comprises a SAM comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and the full length of 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0181] In some embodiments, the viral vector comprises the following SAM, which comprises: (a) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0182] In some embodiments, the viral vector is derived from or based on a DNA virus or an RNA virus.

[0183] In some embodiments, the viral vector is replication-defective, replication-deficient, replication-attenuated, or replication-competent.

[0184] In some embodiments, the viral expression vector is from a virus selected from the group consisting of an adenovirus, an adeno-associated virus, an arenavirus, an alphavirus, a poxvirus, a cytomegalovirus, a rhabdovirus, a vesicular stomatitis virus, a flavivirus, a Maraba virus, and a vaccinia virus.

[0185] In some embodiments, the viral vector is derived from or based on a virus from a taxonomic family selected from the group consisting of: Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia viruses, e.g., Modified Vaccinia Ankara virus (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculoviruses, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphaviruses, e.g., Venezuelan equine encephalitis virus).

[0186] In some embodiments, the viral vector is derived from or based on an arenavirus vector selected from the group consisting of: lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

[0187] In some embodiments, the viral vector is derived from or based on an arenavirus vector selected from: lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus).

[0188] In some embodiments, the arenavirus vector comprises a bi-segmented genome.

[0189] In some embodiments, the arenavirus vector comprises a three-segmented genome.

[0190] In some embodiments, the viral vector is a human adenovirus or a simian adenovirus.

[0191] In some embodiments, the simian adenovirus is selected from the group consisting of a chimpanzee adenovirus, a gorilla adenovirus, and a rhesus adenovirus.

[0192] In some embodiments, the viral vector is derived from or based on an adenovirus vector selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (ChAd), gorilla adenovirus, and rhesus monkey adenovirus.

[0193] In some embodiments, a ChAd is selected from the group consisting of ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22), ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (AdC28), ChAdY27 (AdC28), ChAdY30 (AdC31), ChAdY31 (AdC32), ChAdY32 (AdC33), ChAdY33 (AdC34), ChAdY34 (AdC35), ChAdY35 (AdC36), ChAdY36 (AdC37), ChAdY37 (AdC38), ChAdY38 (AdC39), ChAdY40 (AdC41), ChAdY41 (AdC42), ChAdY42 (AdC43), ChAdY43 (AdC44), ChAdY44 (AdC45), ChAdY45 (AdC46), ChAdY46 (AdC47), ChAdY47 (AdC48), ChAdY48 (AdC49), ChAdY50 (AdC49), ChAdY51 (AdC41), ChAdY52 (AdC43), ChAdY53 (AdC47), ChAdY54 (AdC49), ChAdY61 (AdC49), ChAdY62 (AdC41), ChAdY63 (AdC43), ChAdY71 (AdC49), ChAdY72 (AdC41), ChAdY73 (AdC43), ChAdY74 (AdC45), ChAd Ad30 (AdC30), ChAd31 (AdC31), ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 (AdC55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAd143 (AdC143), ChAd144 (AdC144), ChAd145 (AdC145), and ChAd147 (AdC147).

[0194] In some embodiments, the ChAd is ChAd68.

[0195] In some embodiments, the gorilla adenovirus is selected from GC44, GC45, and GC46.

[0196] In some embodiments, the rhesus adenovirus (RhAd) is selected from RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, and RhAd66.

[0197] In some embodiments, the human adenovirus or simian adenovirus comprises modifications of one or more adenoviral genes.

[0198] In some embodiments, the one or more adenoviral genes are selected from E1, E3, and E4 ORF2-4.

[0199] In some embodiments, the modification comprises deleting or substituting one or more nucleotides in one or more adenoviral genes, or inserting one or more nucleotides into one or more adenoviral genes.

[0200] In some embodiments, the human adenovirus or simian adenovirus comprises a deletion of at least a portion of the El region.

[0201] In some embodiments, the polynucleotide is inserted into a deletion of at least a portion of the El region.

[0202] In some embodiments, the viral vector comprises any one of the nucleic acid sequences SEQ ID NOs: 520-521.

[0203] In some embodiments, the viral vector comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 520-521, wherein the percent identity is across the entire length of the nucleic acid sequence of SEQ ID NOs: 520-521.

[0204] In some embodiments, the viral vector comprises an expression vector comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; and (d) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528; (d) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (e) a polyA sequence; and (f) a fifth polynucleotide sequence comprising nucleotides 4620 to 32350 of SEQ ID NO: 520.

[0205] In some embodiments, the viral vector comprises an expression vector comprising: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (d) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to an amino acid sequence that is any one of SEQ ID NOs: 1-344, wherein the percent identity is across the entire length of SEQ ID NOs: 1-344; (e) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (f) a polyA sequence; and (g) a fifth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520. NO: nucleotides 4620 to 32350 of 520.

[0206] Also disclosed herein are host cells comprising any of the polynucleotides, SAMs, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0207] In some embodiments, the one or more polynucleotides are not integrated into the host cell genome, eg, are episomal.

[0208] In some embodiments, the one or more polynucleotides are integrated into the host cell genome.

[0209] In some embodiments, the host cell is a mammalian cell.

[0210] In some embodiments, the mammalian cell is a human cell.

[0211] In some embodiments, the mammalian cell is not a human cell.

[0212] In some embodiments, the host cell is in vitro.

[0213] In some embodiments, the host cell is in vivo.

[0214] Also disclosed herein is a polypeptide comprising any one of the HIV immunogenic amino acid sequences SEQ ID NOs: 527-528.

[0215] Also disclosed herein are polypeptides comprising an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

[0216] Also disclosed herein are compositions comprising: (a) one or more of any polynucleotide, SAM, expression cassette, expression vector, viral vector, LNP, PNP, or nanoemulsion disclosed herein; and (b) a pharmaceutically acceptable diluent, carrier, or excipient.

[0217] In some embodiments, the composition comprises two or more of any polynucleotide, SAM, expression cassette, expression vector, viral vector, LNP, PNP, or nanoemulsion disclosed herein.

[0218] Also disclosed herein is a composition comprising: (a) one or more of a SAM, expression cassette, LNP, PNP, or nanoemulsion disclosed herein; and (b) one or more expression cassettes or viral vectors disclosed herein.

[0219] Also disclosed herein is a composition comprising: (a) one or more polypeptides disclosed herein; and (b) a pharmaceutically acceptable diluent, carrier, or excipient.

[0220] In some embodiments, the composition comprises two or more polypeptides disclosed herein.

[0221] In some embodiments, any of the compositions disclosed herein further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

[0222] In some embodiments, any of the compositions disclosed herein further comprises one or more of an adjuvant, an immunostimulant, a detergent, a micelle former, and an oil.

[0223] In some embodiments, the immunostimulatory agent is selected from a toll-like receptor (TLR) agonist, a cytokine, a non-coding immunostimulatory polynucleotide, an inhibitor of an inhibitory immune checkpoint protein, or a stimulator of a stimulatory immune checkpoint protein.

[0224] In some embodiments, the cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, GM-CSF, FLT3LG, and combinations and functional variants thereof.

[0225] In some embodiments, the non-coding immunostimulatory polynucleotide is selected from a pathogen activated molecular pattern (PAMP), a cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide, and an immunostimulatory RNA (isRNA, such as CV8102).

[0226] In some embodiments, the composition is formulated for administration via a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, and mucosal (eg, buccal, intranasal, rectal, vaginal).

[0227] In some embodiments, the composition is formulated as a liquid.

[0228] In some embodiments, the composition is lyophilized.

[0229] Also disclosed herein are kits comprising one or more components selected from one or more of the following: a polynucleotide, a SAM, an expression cassette, an expression vector, a viral vector, an LNP, a PNP, a nanoemulsion, or a polypeptide disclosed herein.

[0230] Also disclosed herein are kits comprising: (a) a first component selected from any one of the SAMs, LNPs, PNPs, or nanoemulsions disclosed herein; and (b) a second component comprising any one of the expression vectors or viral vectors disclosed herein.

[0231] Also disclosed herein are kits comprising one or more unit doses of any of the following: a polynucleotide, a SAM, an expression cassette, an expression vector, a viral vector, an LNP, a PNP, a nanoemulsion, or a polypeptide disclosed herein.

[0232] Also disclosed herein are kits comprising: (a) one or more unit doses of any of the SAMs, LNPs, PNPs, or nanoemulsions disclosed herein; and (b) one or more unit doses of any of the expression vectors or viral vectors disclosed herein.

[0233] Also disclosed herein are kits comprising one or more unit doses of any of the polypeptides disclosed herein.

[0234] In some embodiments, one or more components are in a single container.

[0235] In some embodiments, one or more components are in two or more separate containers.

[0236] In some embodiments, the first component and the second component are in a single container.

[0237] In some embodiments, the first component and the second component are in two or more separate containers.

[0238] In some embodiments, one or more unit doses are in a single container.

[0239] In some embodiments, one or more unit doses are in two or more separate containers.

[0240] In some embodiments, the one or more containers are selected from the group consisting of a vial, an ampoule, and a prefilled syringe.

[0241] In some embodiments, one or more containers contain one or more SAMs, one or more LNPs, one or more PNPs, one or more nanoemulsions, one or more carriers, or one or more polypeptides in an aqueous solution.

[0242] In some embodiments, one or more unit doses are identical.

[0243] In some embodiments, one or more unit doses are different.

[0244] In some embodiments, a unit dose of one or more viral vectors is about 10 3 to about 10 15 The amount of virus expressed is in the range of focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or virus particles (vp).

[0245] In some embodiments, a unit dose of one or more viral vectors is about 10 4 to about 10 7 Virus FFU or PFU or IU or vp.

[0246] In some embodiments, a unit dose of one or more viral vectors is about 10 3 to about 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 or 10 15 Virus FFU or PFU or IU or vp.

[0247] In some embodiments, the unit dose of one or more SAMs ranges from about 1 μg to about 1000 μg.

[0248] In some embodiments, the unit dose of one or more SAMs is from about 15 μg to about 500 μg.

[0249] In some embodiments, the unit dose of one or more SAMs is from about 1 μg to about 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg.

[0250] In some embodiments, any of the compositions or kits disclosed herein further comprises one or more unit doses of one or more additional therapeutic agents.

[0251] In some embodiments, the one or more additional therapeutic agents are selected from one or more agents that activate latent HIV, one or more agonists or activators of one or more toll-like receptors (TLRs), one or more interleukin receptor agonists, one or more cytokines, one or more receptor agonists, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors, one or more agonists, activators or stimulators of T cell stimulatory immune checkpoint proteins or receptors, one or more CD47 inhibitors, one or more immune-based therapies, one or more broadly neutralizing antibodies (bnAbs), and one or more antiviral agents.

[0252] In some embodiments, the one or more agents that activate latent HIV are one or more latency reversing agents (LRAs).

[0253] In some embodiments, the one or more LRAs are selected from the group consisting of: an agonist or activator of one or more toll-like receptors (TLRs), a histone deacetylase (HDAC) inhibitor, a proteasome inhibitor, a protein kinase C (PKC) activator, a Smyd2 inhibitor, a BET-bromodomain 4 (BRD4) inhibitor, ionomycin, an inhibitor of apoptosis protein (IAP) antagonist, and a second mitochondrial-derived caspase activator (SMAC) mimetic.

[0254] In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0255] In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620 (visamod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod), and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688, R848 (resiquimod), CV8102 (dual TLR7 / TLR8 agonist), and NKTR-262 (dual TLR7 / TLR8 agonist).

[0256] In some embodiments, any of the compositions or kits disclosed herein further comprises GS 9620 (visamod).

[0257] In some embodiments, the TLR9 agonist is selected from the group consisting of AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, linimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN-1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042.

[0258] In some embodiments, any of the compositions or kits disclosed herein further comprises lefetomod (MGN-1703).

[0259] In some embodiments, the interleukin is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG.

[0260] In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG, and combinations and functional variants thereof.

[0261] In some embodiments, the receptor agonist is an agonist of one or more receptors selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain-containing protein 2 (NOD2).

[0262] In some embodiments, the one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors inhibit a checkpoint protein or receptor selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated protein 4). off (BTLA); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1).

[0263] In some embodiments, one or more agonists, activators or stimulators of a T cell stimulatory immune checkpoint protein or receptor agonizes, activates or stimulates a T cell stimulatory immune checkpoint protein or receptor selected from the group consisting of: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155).

[0264] In some embodiments, the CTLA4 inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, C G-0161, ATOR-1144, PBI-5D3H5, FPT-155(CTLA4 / PD-L1 / CD28), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA 4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA4 / PD-1) and BPI-002.

[0265] In some embodiments, the PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cemiplizumab, pidilizumab, AB122 (sepalizumab), AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, PF-0680 1591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genoluzumab (CBT-501), LZM-009, BCD-100 , LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalizumab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP -3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-0 13(PD-1 / LAG-3), FS-118(LAG-3 / PD-L1)MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), MED I-5752(CTLA4 / PD-1), RO-7121661(PD-1 / TIM-3), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA 4 / PD-1), M7824 (PD-L1 / TGFb-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3 415244(TIM3 / PDL1), INBRX-105(4-1BB / PDL1), GS-4224, GS-4416, INCB086550 and MAX10181.

[0266] In some embodiments, the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors.

[0267] Also disclosed herein are methods for eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof. In some embodiments, the method comprises administering to the subject any one or more of the polynucleotides, SAMs, LNPs, PNPs, nanoemulsions, expression cassettes, expression vectors, viral vectors, or polypeptides disclosed herein, or any combination thereof.

[0268] Also disclosed herein are methods for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof. In some embodiments, the method comprises administering to the subject any one or more of the polynucleotides, SAMs, LNPs, PNPs, nanoemulsions, expression cassettes, expression vectors, viral vectors, or polypeptides disclosed herein, or any combination thereof.

[0269] In some embodiments, the subject is infected with HIV-1, suspected of being infected with HIV-1, or at risk of being infected with HIV-1.

[0270] In some embodiments, the subject is chronically infected with HIV-1.

[0271] In some embodiments, the subject is acutely infected with HIV-1.

[0272] In some embodiments, the subject has Fiebig stage IV or earlier (eg, Fiebig stage III, Fiebig stage II, or Fiebig stage I) HIV-1 infection.

[0273] In some embodiments, administering comprises a route of administration selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (eg, buccal, intranasal, rectal, vaginal).

[0274] In some embodiments, the method comprises administering about 10 3 to about 10 15 One or more viral vectors are administered in a dosage range of 100 viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp).

[0275] In some embodiments, one or more viral vectors are administered at a dose of about 10 4 to about 10 7 Virus FFU or PFU or IU or vp, for example about 103 to about 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 or 10 15 The dosage range is 100 virus FFU or PFU or IU or vp.

[0276] In some embodiments, the method comprises administering one or more SAMs, LNPs, PNPs, or nanoemulsions disclosed herein at a dose range of about 1 μg to about 1000 μg.

[0277] In some embodiments, one or more SAMs, LNPs, PNPs, or nanoemulsions are administered in a dose range of about 15 μg to about 500 μg.

[0278] In some embodiments, one or more SAMs, LNPs, PNPs, or nanoemulsions are administered in a dose range of about 1 μg to about 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg.

[0279] In some embodiments, any of the methods disclosed herein comprises a prime-boost regimen comprising: (a) administering a priming composition at a first time point followed by administering one or more boosting compositions at one or more subsequent time points (e.g., prime-boost-boost-boost, etc.); (b) administering a priming composition at a first time point followed by administering a boosting composition at a second time point (e.g., prime-boost-prime-boost, etc.) in one or more iterations; or (c) administering a priming composition at a first time point, administering a boosting composition at one or more subsequent time points, and administering a priming composition at a subsequent time point after administering the boosting composition in one or more iterations (e.g., prime-boost-boost-prime, etc.).

[0280] In some embodiments, the interval between administration of the priming composition and the one or more boosting compositions is at least 1 week, 2 weeks, 3 weeks, or 1 month, such as at least 2 months, 3 months, 4 months, 5 months, or 6 months.

[0281] In some embodiments, the priming composition and the boosting composition comprise polynucleotides encoding the same immunogenic polypeptide.

[0282] In some embodiments, the priming composition and the boosting composition comprise polynucleotides encoding different immunogenic polypeptides.

[0283] In some embodiments, the priming composition and the boosting composition comprise the same one or more polypeptides, polynucleotides, SAMs, LNPs, PNPs, nanoemulsions, or viral vectors.

[0284] In some embodiments, the priming composition and the boosting composition comprise different polypeptides, polynucleotides, SAMs, LNPs, PNPs, nanoemulsions, or viral vectors.

[0285] In some embodiments, any of the methods disclosed herein comprises priming with a first polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector, followed by boosting with a second polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector.

[0286] In some embodiments, the prime-boost regimen comprises: (i) priming with a viral expression vector and then boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA; (ii) priming with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA, and then boosting with a viral expression vector; (iii) priming with a first viral expression vector and then boosting with a second viral expression vector, wherein the first viral expression vector and the second viral expression vector are from the same, related, or unrelated taxonomic families; (iv) priming with a first replication-deficient viral expression vector and then boosting with a second replication-deficient viral expression vector, wherein the first replication-deficient viral expression vector and the second replication-deficient viral expression vector are from the same, related, or unrelated taxonomic families; (v) priming with a first attenuated defective viral expression vector and then boosting with a second replication-attenuated viral expression vector, wherein the first replicating attenuated viral expression vector and the second replicating attenuated viral expression vector are from the same, related or unrelated taxonomic family; (vi) priming with a replication-deficient viral expression vector and then boosting with a replicating attenuated viral expression vector; (vii) priming with a replication-deficient viral expression vector and then boosting with a replication-deficient viral expression vector; (viii) priming with a viral vector and then boosting with a composition comprising a SAM; (ix) priming with an adenoviral viral expression vector and then boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA; (x) priming with an adenoviral vector and then boosting with a composition comprising a SAM; (xi) priming with a ChAd expression vector and then boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA; or (xii) priming with a ChAd vector and then boosting with a composition comprising a SAM.

[0287] In some embodiments, the subject is not receiving antiretroviral therapy (ART), or is discontinued from ART prior to administration of the one or more polynucleotides, polypeptides, SAMs, LNPs, PNPs, nanoemulsions, expression cassettes, or viral vectors.

[0288] In some embodiments, ART is discontinued after one or more administrations of the polynucleotide, polypeptide, SAM, LNP, PNP, nanoemulsion, expression cassette, or viral vector.

[0289] In some embodiments, any of the methods disclosed herein further comprises administering to the subject one or more additional therapeutic agents, eg, two, three, four, or more additional therapeutic agents.

[0290] In some embodiments, the one or more therapeutic agents are selected from one or more agents that activate latent HIV, one or more toll-like receptor (TLR) agonists or activators, one or more interleukin receptor agonists, one or more cytokines, one or more receptor agonists, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors, one or more agonists, activators or stimulators of T cell stimulatory immune checkpoint proteins or receptors, one or more CD47 inhibitors, one or more antiviral agents, one or more immune-based therapies, and one or more broadly neutralizing antibodies (bnAbs).

[0291] In some embodiments, the one or more agents that activate latent HIV are one or more latency reversing agents (LRAs).

[0292] In some embodiments, the one or more LRAs are selected from: agonists or activators of one or more toll-like receptors (TLRs), histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitor of apoptosis protein (IAP) antagonists, and second mitochondrial-derived activator of caspase (SMAC) mimetics.

[0293] In some embodiments, the one or more TLR agonists or activators are selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0294] In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620 (visamod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod), and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688, R848 (resiquimod), CV8102 (dual TLR7 / TLR8 agonist), and NKTR-262 (dual TLR7 / TLR8 agonist).

[0295] In some embodiments, any of the methods disclosed herein further comprises administering GS 9620 (visamod) to the subject.

[0296] In some embodiments, the TLR9 agonist is selected from the group consisting of AST-008, cobitomot, CMP-001, IMO-2055, IMO-2125, linimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatomod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefetomod (MGN-1703), CYT-003, CYT-003-QbG10, tesotommod, and PUL-042.

[0297] In some embodiments, any of the methods disclosed herein further comprises administering fetomod (MGN-1703) to the subject.

[0298] In some embodiments, the one or more bnAbs are HIV bnAbs.

[0299] In some embodiments, any of the methods disclosed herein further comprises administering one or more bnAbs.

[0300] In some embodiments, HIV bnAbs are highly effective against most circulating HIV strains, neutralize a wide range of genetically diverse HIV-1 subtypes, and / or potently neutralize a substantial proportion of primary isolates, or exhibit some clade-spanning ability, but are less effective at neutralizing tier 2 and tier 3 viruses.

[0301] In some embodiments, bnAbs are directed to the surfaces of Env: the CD4 binding site (CD4bs), N-glycans associated with the V1 / V2 and V3 loops, the silent face of gp120, the membrane proximal external region (MPER) of gp41, or a larger site spanning the interface between gp41 and gp120.

[0302] In some embodiments, the bnAb is selected from 10-1074, 10E8, 12A12, 12A21, 2F5, 2G12, 3BC176, 3BNC117, 3BNC55, 3BNC60, 3BNC62, 447-52D, 4E10, 5H / I1-BMV-D5, 8ANC195, b12, CHO1, CH02, CH03, CH04, CH103, HGN194, HJ16, HK20, M66.6, NIH45-46, PG16, PG9, PGT121, PGT122, PGT123, PGT125, PGT126, PGT127, PGT128, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, P GT144, PGT145, VRC-CH30, CRC-CH31, CRC-CH32, CRC-CH33, CRC-CH34, VRC-PG04, VRC-PG04b, VRC01, VRC02, VRC03 and Z13.

[0303] In some embodiments, the bnAb is an anti-CD4 bnAb.

[0304] In some embodiments, the anti-CD4 bnAb is selected from 3BNC117 and VRC01.

[0305] In some embodiments, the bnAb is an anti-V3 bnAb.

[0306] In some embodiments, the anti-V3 bnAb is 10-1074.

[0307] In some embodiments, the one or more interleukin receptor agonists are agonists of an interleukin selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, and FLT3LG.

[0308] In some embodiments, the one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG, and combinations and functional variants thereof.

[0309] In some embodiments, the one or more receptor agonists are agonists of a receptor selected from the group consisting of: fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain-containing protein 2 (NOD2).

[0310] In some embodiments, the one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors are inhibitors of T cell inhibitory immune checkpoint proteins or receptors selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocytes); cell-associated (BTLA); PVR-associated immunoglobulin domain-containing (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1).

[0311] In some embodiments, one or more agonists, activators or stimulators of a T cell stimulatory immune checkpoint protein or receptor agonizes, activates or stimulates a T cell stimulatory immune checkpoint protein or receptor selected from the group consisting of: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155).

[0312] In some embodiments, the CTLA4 inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, C G-0161, ATOR-1144, PBI-5D3H5, FPT-155(CTLA4 / PD-L1 / CD28), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA 4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA4 / PD-1) and BPI-002.

[0313] In some embodiments, the PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplizumab, pidilizumab, AB122 (sepalizumab), AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, M GA-012, BI-754091, AGEN-2034 (batilizumab), JS-001 (toripalimab), JNJ-63723283, genoluzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalimumab), MSB-2311, JTX-4014, BGB-A333, SHR-131 6. CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), MGD-019 (PD-1 / CTLA4), KN-046 (PD- 1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFb-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), INBRX-105 (4-1BB / PDL1), GS-4224, GS-4416, INCB086550, and MAX10181.

[0314] In some embodiments, the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors.

[0315] In some embodiments, the subject remains symptom-free of HIV and AIDS for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or longer without antiretroviral therapy (ART) following one or more administrations of one or more of the polynucleotide, polypeptide, SAM, LNP, PNP, microemulsion, expression cassette, or viral vector, optionally in combination with one or more additional therapeutic agents.

[0316] In some embodiments, following one or more administrations of one or more of the polynucleotide, polypeptide, SAM, LNP, PNP, microemulsion, expression cassette, or viral vector, optionally in combination with one or more additional therapeutic agents, the subject has a viral load of less than 500 copies per ml of blood, e.g., less than 400, less than 300, less than 200, less than 100, less than 50, for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or longer, in the absence of antiretroviral therapy (ART).

[0317] Also disclosed herein is the use of any of the polynucleotides, SAMs, expression cassettes, LNPs, PNPs, nanoemulsions, expression vectors, viral vectors, polypeptides, or compositions disclosed herein for eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof.

[0318] Also disclosed herein is the use of any of the polynucleotides, SAMs, expression cassettes, LNPs, PNPs, nanoemulsions, expression vectors, viral vectors, polypeptides, or compositions disclosed herein for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof.

[0319] Also disclosed herein is the use of any of the polynucleotides, SAMs, expression cassettes, LNPs, PNPs, nanoemulsions, expression vectors, viral vectors, polypeptides, or compositions disclosed herein as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0320] Figure 1 Presents an 8-step workflow for designing fusion peptides to elicit an antiviral response.

[0321] Figure 2 Representative methods demonstrating population-based vaccine construction approaches.

[0322] Figure 3The steps of the Conservative Walking Analysis (CWA) algorithm as described herein are shown.

[0323] Figures 4A to 4B . Figure 4A It is demonstrated how "bivalent conservation" can be determined based on the prevalence of the two most common 9-mers among all considered viral sequences in a population. Figure 4A SEQ ID NOs: 475-476, 476-477, 476, 476, 478-479, and 479-480 are disclosed in the figure above, respectively, in order of appearance. Figure 4A Also disclosed in the figure below are "QNLQGQMVH" as SEQ ID NO: 481, "QNIQGQMVH" as SEQ ID NO: 482, and "PNIQGQMVH" as SEQ ID NO: 483. Figure 4B Demonstrates how to identify conserved regions based on the distribution of "bivalent conservation" among 9-mer positions. HIV-1 Gag p24 is used as a representative protein.

[0324] Figures 5A to 5C . Figure 5A Unique 9-mers extracted from aligned natural sequences are shown. Figure 5B Shows a directed acyclic graph constructed based on 9-mer pairs of nodes and their connections. Figure 5C Displays the connection patterns of the 9-mers in a 9-mer pair. When there are two choices for connection, the final connection is determined by the prevalence of each connection in naturally occurring sequences. Figures 5A to 5C It is disclosed that "IIIIIIIIR" is SEQ ID NO: 467, "GIIIIIIIIH" is SEQ ID NO: 473, "AIIIIIIIIK" is SEQ ID NO: 474, "GIIIIIIIIR" is SEQ ID NO: 484, "GIIIIIIIII" is SEQ ID NO: 485, "AIIIIIIIII" is SEQ ID NO: 486, "IIIIIIIIIK" is SEQ ID NO: 487 and "IIIIIIIIIH" is SEQ ID NO: 488.

[0325] Figure 6 Presenting the results of human proteome cross-recognition analysis. Figure 6 The "HIV peptide" sequence is disclosed as SEQ ID NO: 492, and the "human protein 9-mer" sequence is disclosed as SEQ ID NOs: 493-498 and 493, respectively, in the order of appearance.

[0326] Figure 7 Demonstrates how polypeptide fragment alignment analysis can reduce or eliminate the potential presentation of deleterious or undesirable epitopes in joining regions.

[0327] Figure 8 A rational antiviral immunogen design approach that considers inter- and intra-patient viral sequence diversity and host MHC class I and class II molecule binding and T cell recognition is presented. The approach and resulting immunogens are exemplified herein by immunogens that elicit human immune responses to HIV-1.

[0328] Figure 9 Schematic diagram showing antigen-specific T cell responses in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02 (corresponding to SEQ ID NOs: 520-523, respectively), or PBS control.

[0329] 10A to 10D Schematic diagram showing antigen-specific T cell responses in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02, or PBS control. Figure 10A T cell responses to Pol-1 in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02, or PBS control are shown. Figure 10B T cell responses to Pol-2 in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02, or PBS control are shown. Figure 10C T cell responses to Gag in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02, or PBS control are shown. Figure 10D T cell responses to Nef in mice vaccinated with ChAd-01, ChAd-02, SAM-01, SAM-02, or PBS control are shown.

[0330] Figure 11 Schematic diagram showing the design, procedures, and treatments of a clinical trial.

[0331] Figure 12 Schematic representations of the fusion polypeptides designated Immunogen 1A and Immunogen 1B, which correspond to the amino acid sequences of SEQ ID NOs: 527-528, respectively, are shown. The starting and ending sites of the peptide fragments of Gag, Pol, and Nef are indicated in the alignment details.

[0332] Figure 13Figure 2 shows SIV-specific T cell responses in rhesus macaques following vaccination with ChAd / samRNA alone (circles) or in combination with anti-PD-1 antibodies (squares), anti-CTLA4 antibodies (triangles), or FLT3R agonists (inverted triangles). IFNγ ELISpot, sum of Env, Pol, and Gag overlapping peptide pools, background subtracted. Mean ± SEM. Black arrows represent ChAd immunizations, gray arrows represent samRNA immunizations.

[0333] Figure 14A A linear schematic diagram of the ChAd vector containing the CMV promoter, immunogen and polyA is shown.

[0334] Figure 14B A linear schematic diagram of the samRNA vector containing the 26S subgenomic promoter, immunogen, and polyA is shown.

[0335] Figure 15 Shown is the breadth of SIV Gag-specific IFNγ T cell responses in PBMCs from rhesus macaques following vaccination with ChAd / samRNA alone or in combination with anti-PD-1 antibody (aPD1), anti-CTLA4 antibody (aCTLA4), or FLT3R agonist (FLT3Ra).

[0336] Figure 16 Shown are SIV-specific CD8+ and CD4+ T cell populations in rhesus macaques following vaccination with ChAd / samRNA alone or in combination with anti-PD-1 antibody (aPD1), anti-CTLA4 antibody (aCTLA4), or FLT3R agonist (FLT3Ra), as measured by intracellular cytokine staining of PBMCs following overnight stimulation with four overlapping peptide pools spanning Gag, Pol, and Env, with background subtraction, at week 14; pool sums are presented as median (IQR).

[0337] Figure 17 Shown is the function of SIV-specific T cells in rhesus macaques following vaccination with ChAd / samRNA alone or in combination with anti-PD-1 antibody (aPD1), anti-CTLA4 antibody (aCTLA4), or FLT3R agonist (FLT3Ra), as measured by intracellular cytokine staining of PBMCs after overnight stimulation at week 14 with background-subtracted pools of four overlapping peptides spanning Gag, Pol, and Env.

[0338] Figure 18 Shown are serum exposure levels of aPD-1, aCTLA4, and Flt3Ra in rhesus monkeys.

[0339] Figure 19 Shown are PD-1 receptor occupancy in rhesus macaques following combination vaccination with ChAd / samRNA and anti-PD-1 antibody (aPD1) administered by SC or IV route compared to vaccination alone.

[0340] Figure 20 Shown is the expansion of cDC1 cells from baseline over time in PBMCs of rhesus macaques dosed with a FLT3R agonist (FLT3Ra) followed by vaccination with ChAd / samRNA. DETAILED DESCRIPTION

[0341] HIV immunogenic polypeptides are provided herein. In some embodiments, the HIV immunogenic polypeptide comprises the amino acid sequence of SEQ ID NOs: 527-528.

[0342] Also provided herein are polynucleotides encoding HIV immunogenic polypeptides. In some embodiments, these polynucleotides comprise a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524-526. In some embodiments, these polynucleotides comprise a nucleic acid sequence encoding any of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528. In some embodiments, these polynucleotides comprise a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528, wherein the percent identity spans the entire length of the nucleic acid sequence encoding the polypeptide of any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

[0343] Also disclosed herein are expression cassettes comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein. In some embodiments, the expression cassette comprises a polynucleotide encoding any of the HIV immunogenic polypeptides of SEQ ID NOs: 1-371, 373-377, 407-411, 422-423, 430-435, 527, and 528. In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any of SEQ ID NOs: 524-526.

[0344] Also disclosed herein are expression vectors comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein. In some embodiments, the expression vector comprises a polynucleotide encoding any of the HIV immunogenic polypeptides SEQ ID NOs: 1-371, 373-377, 407-411, 422-423, 430-435, 527, and 528. In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any of SEQ ID NOs: 520-521. In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 522-523. In some embodiments, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 524-526.

[0345] Also disclosed herein are self-amplifying mRNAs (SAMs) comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein. In some embodiments, the SAM comprises a polynucleotide encoding any of the HIV immunogenic polypeptides SEQ ID NOs: 1-371, 373-377, 407-411, 422-423, 430-435, 527, and 528. In some embodiments, the SAM comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any of SEQ ID NOs: 522-523. In some embodiments, the SAM comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 524-526.

[0346] Also disclosed herein are viral vectors comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein. In some embodiments, the viral vector comprises a polynucleotide encoding any of the HIV immunogenic polypeptides SEQ ID NOs: 1-371, 373-377, 407-411, 422-423, 430-435, 527, and 528. In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleic acid sequence of any of SEQ ID NOs: 520-521. In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence of any one of SEQ ID NOs: 524-526.

[0347] Also disclosed herein are lipid nanoparticles (LNPs), polymeric nanoparticles (PNPs), or nanoemulsions comprising any of the polynucleotides, expression cassettes, expression vectors, or viral vectors disclosed herein.

[0348] Also disclosed herein are cells comprising any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0349] Also disclosed herein are compositions comprising any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0350] Also disclosed herein are kits comprising any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0351] Also disclosed herein are methods of eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0352] Also disclosed herein are methods of using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein to treat or prevent human immunodeficiency virus (HIV) in a subject in need thereof.

[0353] Also disclosed herein are methods of using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein to treat or prevent human immunodeficiency virus (HIV) in a subject in need thereof.

[0354] HIV immunogenic peptides

[0355] HIV immunogenic polypeptides are disclosed herein. As used herein, the phrase "HIV immunogenic polypeptide" refers to a polypeptide that can elicit an immunogenic response against human immunodeficiency virus (HIV-1) in a subject in need thereof. As used herein, an "immunogen" is a substance, such as an antigen, that elicits or is capable of eliciting an immune response. The phrases "HIV immunogenic polypeptide" and "fusion polypeptide" are used interchangeably.

[0356] Peptide fragments

[0357] HIV immunogenic polypeptides comprise multiple polypeptides or peptide fragments encoded by one or more HIV-1 genes. A "fragment" of an HIV immunogenic polypeptide (e.g., a fusion polypeptide) as described herein is a contiguous sequence comprising at least 8 amino acids relative to a reference sequence (e.g., the HIV-1 HXB2 reference sequence for the Env, Gag, Nef, and Pol polypeptides, provided herein as SEQ ID NOs: 403-406, respectively). The HIV immunogenic polypeptides described herein can be described as "fusion" polypeptides because they are assembled from linked or tandem polypeptide or peptide fragments of two or more HIV-1 proteins. With respect to the HIV-1 protein reference sequence, the polypeptide or peptide fragment can correspond to discontinuous sequences of the same HIV-1 protein or different HIV-1 proteins. Generally, HIV immunogenic polypeptides (e.g., fusion polypeptides) are non-naturally occurring and can be synthetically or recombinantly produced.

[0358] With respect to HIV-1 genes encoding polypeptide fragments for assembling the fusion polypeptides described herein, in various embodiments, the fusion polypeptide comprises a plurality of polypeptide fragments of one or more human immunodeficiency virus-1 (HIV-1) proteins encoded by one or more (e.g., two or more, three or more, four or more) HIV-1 genes selected from Gag, Nef, Env, Pol, Vpu, Vpr, and Vif (e.g., two or more, three or more, four or more HIV-1 genes selected from Gag, Nef, Env, Pol, and Vif). In some embodiments, the plurality of polypeptide fragments consists solely of polypeptide fragments encoded by HIV-1 genes Env, Gag, Nef, and Pol, e.g., polypeptide fragments encoded by HIV-1 Tat, Rev, Vif, Vpr, and / or Vpu genes are not included. In some embodiments, the plurality of polypeptide fragments consists solely of polypeptide fragments encoded by HIV-1 genes Gag, Nef, and Pol, e.g., polypeptide fragments encoded by HIV-1 Env, Tat, Rev, Vif, Vpr, and / or Vpu genes are not included. In some embodiments, the plurality of polypeptide fragments consist only of polypeptide fragments encoded by HIV-1 genes Gag and Nef, for example, and do not include polypeptide fragments encoded by HIV-1 Env, Pol, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide fragments consist only of polypeptide fragments encoded by HIV-1 genes Pol and Nef, for example, and do not include polypeptide fragments encoded by HIV-1 Env, Gag, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide fragments consist only of polypeptide fragments encoded by HIV-1 genes Pol and Env, for example, and do not include polypeptide fragments encoded by HIV-1 Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide fragments consist only of polypeptide fragments encoded by HIV-1 Pol gene, for example, and do not include polypeptide fragments encoded by HIV-1 Env, Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes. In various embodiments, the plurality of polypeptide fragments does not contain fragments encoded by one, two, three, or four of the HIV Tat, Rev, Vif, Vpr, and / or Vpu genes.

[0359] With respect to the number of polypeptide fragments assembled, linked, connected, or concatenated into a single fusion polypeptide, in various embodiments, the fusion polypeptide is composed of at least 5 and at most 40 polypeptide fragments (e.g., 5 polypeptide fragments and at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 polypeptide fragments). As the case may be, the polypeptide fragments can be arranged in the same or different order as in the naturally occurring protein.

[0360] With respect to the polypeptide region encoded by an HIV-1 gene that is selected as the polypeptide segment to be included in the fusion polypeptide, in various embodiments, the polypeptide segment is derived from a conserved region in a population of viral proteome sequences. In some embodiments, the conserved region is greater than 80%, e.g., greater than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% conserved across HIV-1 species, e.g., as determined in an inter-patient and / or intra-patient population. As used herein, a conserved region in a polypeptide encoded by an HIV-1 gene refers to the percentage of sequences in a population of sequences that contain an amino acid segment or subsequence (e.g., a segment or 9-mer of 9 amino acids in length) that is identical to the most prevalent amino acid segment or subsequence at a predetermined amino acid segment or subsequence position, wherein the amino acid segment or subsequence position is determined relative to a reference sequence (e.g., an HIV-1 HXB2 polypeptide sequence, e.g., SEQ ID NOs: 403-406). In various embodiments, the conserved region is conserved between one or more HIV-1 clades within group M (e.g., one or more HIV-1 clades AK, e.g., one or more clades A, B, C, D, and G) (e.g., between HIV-1 group M clade B and its recombinant forms (e.g., CRF01_AE)). In some embodiments, the plurality of polypeptide fragments includes at least 2 polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NOs: 1-344, e.g., the polypeptide fragments identified in Table 2.In some embodiments, the plurality of polypeptide fragments comprises at least 2 polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NO: NO: 2, 3, 8, 9, 13, 14, 17, 18, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 62, 63, 64, 65, 66, 67, 68, 69, 72, 73, 74, 75, 76, 77, 80, 81, 82, 83, 84, 85, 86, 87 ,88,89,92,93,101,102,103,104,109,110,115,116,125,126,127,128,129,130,131,132,133,134,135,136,139,140,141,142,143,144,145,146,147,148,149,150,155,156,157,158,159,160,166,167,168,169,170 , 171, 174, 175, 178, 179, 180, 181, 182, 183, 184, 185, 193, 194, 195, 196, 197, 198, 199, 200, 203, 204, 205, 206, 207, 208, 213, 214, 221, 222, 236, 237, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263 , 264, 266, 267, 268, 269, 270, 271, 272, 273, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 305, 306, 307, 308, 309, 310, 313, 314, 315, 316, 317, 318, 321 and 322, e.g., polypeptide fragments identified in Table 3.The start and end positions are relative to the HIV-1 HXB2 reference polypeptide (GenBank Accession No. K03455, ncbi.nlm.nih.gov / nuccore / K03455, provided herein as SEQ ID NOs: 403-406 and identified in Table 1).

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382] With respect to the range of lengths of individual polypeptides or peptide fragments, in various embodiments, each polypeptide fragment is at least 8 amino acids in length and up to about 250 amino acids in length, e.g., at least 8 amino acids in length and up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. In various embodiments, each polypeptide fragment is at least 8 amino acids in length and is up to about 35 amino acids in length, e.g., at least 8 amino acids in length and up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, or 35 amino acids in length. In various embodiments, each polypeptide fragment is at least 15 amino acids in length and is up to about 30 amino acids in length, e.g., at least 15 amino acids in length and up to 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0383] With respect to the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full length of the fusion polypeptide comprises at least about 350 amino acids and at most about 1000 amino acids, e.g., at least about 350 amino acids and at most about 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980 940, 950, 960, 970, 980, 990, or 1000 amino acids. With respect to the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full length of the fusion polypeptide comprises at least about 350 amino acids and at most about 800 amino acids, e.g., at least about 350 amino acids and at most about 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 amino acids. In some embodiments, the total length of the fusion polypeptide is no longer than 800 amino acids, e.g., no longer than 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, 705, or 700 amino acids.

[0384] Generally, fusion polypeptides are immunogenic because they can cause, for example, an immune response to HIV-1 in the human body. In some embodiments, for example, fusion polypeptides as described herein optionally in combination with one or more additional therapeutic agents can cause protection or effective immune response to HIV-1 in the human body, for example, can prevent HIV-1 infection in uninfected individuals or in a treatment environment, can cause an immune response sufficient to induce immune-mediated control of HIV-1 or eradicate HIV-1 in infected individuals. The immunogenicity of fusion polypeptides can be assessed and demonstrated in vitro and in vivo assays as described herein. For example, the immunogenicity of fusion polypeptides can be demonstrated by in vitro assays (including CD4+ and / or CD8+ T cell activation (for example, including cytokine expression and target killing assays) or proliferation assays). T cells can be activated by being exposed to antigen presenting cells (APCs) (for example dendritic cells, for example monocyte-derived dendritic cells) transfected with polynucleotides encoding fusion polypeptides. Such assays are known in the art and described herein. The immunogenicity of the fusion polypeptide can also be demonstrated in an in vivo animal model, for example, by administering a transgenic product of one or more human HLA molecules (available from Jackson Laboratories or Taconic) to mice or non-human primates and assessing CD4+ and / or CD8+ T cell activation (e.g., including serum cytokine levels) or proliferation. In various embodiments, one, two, three, or more of each polypeptide fragment comprises or consists of one or more predicted T cell epitopes, for example, as determined computationally or experimentally. In some embodiments, the fusion polypeptide comprises one or more polypeptide fragments that bind to or are presented by one or more human HLA class I and / or class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), for example, within a single subject or across multiple subjects. In some embodiments, the fusion polypeptide comprises one or more polypeptide fragments that bind to or are presented by at least the human A*0201 HLA class I molecule. In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 8-mer, 9-mer and / or 10-mer polypeptide fragments that bind to or are presented by one or more human HLA class I and / or class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., in a single subject.In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more polypeptide fragments, each 15 to 30 amino acids in length, which are processed intracellularly and presented by one or more human HLA class I and / or class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., in a single subject.

[0385] Tandem peptide fragments

[0386] As the case may be, one or more polypeptide fragments can be directly adjacent to or fused to adjacent fragments, or can be bound, connected or linked to adjacent fragments by one or more peptide linkers. In various embodiments, one or more peptide linkers are selected from one or more of the following: for example, polyalanine linkers, polyglycine linkers, cleavable linkers, flexible linkers, rigid linkers, Nef linking sequences, and combinations thereof within a linker or within a full-length fusion polypeptide. Exemplary fusion protein linkers that can be used to connect one or more polypeptide fragments in the fusion polypeptide of the present invention are described, for example, in Chen et al., Adv Drug Deliv Rev. (2013) 65 (10): 1357-1369. In some embodiments, the polyalanine linker comprises or consists of 2 or 3 consecutive alanine residues, such as AA, AAA, AAY, or AAX, where X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y). In some embodiments, a polyglycine linker is used, such as GGS, GSG, or GGGS (SEQ ID NO: 421).

[0387] In some embodiments, the cleavable linker is selected from a 2A cleavable peptide. Exemplary 2A cleavable peptides that can be used to link one or more polypeptide fragments in the fusion polypeptide of the present invention are described, for example, in Donnelly et al., J. Gen. Virol (2001), 82, 1027-1041 and Chng et al., mAbs (2015) 7: 2, 403-412. Exemplary cleavable peptides that can be used to link one or more polypeptide fragments include, but are not limited to, 2A cleavage sequences (e.g., foot-and-mouth disease virus (F2A), equine rhinitis virus A (E2A), porcine teschovirus-1 (P2A), and scutellaria avium virus (T2A)) and furin recognition / cleavage sequences (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)). In certain embodiments, the furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)) is combined or fused in a single linker with a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and scutellaria avium virus (T2A)). See, e.g., Chng et al., mAbs (2015) 7: 2, 403-412. In various embodiments, the 2A cleavable linker comprises, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388); or comprises the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388). NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388), or consists of the amino acid sequence.As appropriate, in certain embodiments, the furin recognition / cleavage sequence may be located at the N-terminus or C-terminus of the 2A linker. In some embodiments, the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382), and RRKR (SEQ ID NO: 383). REKR (SEQ ID NO: 382) is a naturally occurring cleavable linker in HIV and SIV envelope glycoprotein precursors (Bahbouhi et al., Biochem. J. (2002) 366, 863-872). In some embodiments, the fusion polypeptide comprises one or more Nef linker sequences comprising or consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), or GALDI (SEQ ID NO: 391); or comprises or consists of an amino acid sequence selected from VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390), and GALDI (SEQ ID NO: 391). Exemplary linkers that can be used to join or connect one or more polypeptide fragments in a fusion polypeptide are provided in Table 4.

[0388]

[0389] A polypeptide fragment encoded by the HIV-1 Gag gene

[0390] In various embodiments, the fusion polypeptide comprises one or more viral proteins encoded by the HIV-1 Gag gene, or one or more fragments thereof, of a fragment or subsequence thereof. In some embodiments, the one or more viral proteins encoded by the HIV-1 Gag gene are selected from p17 (N-terminal matrix), p24 (capsid), p7 (nucleocapsid), and p6 (C-terminal). In some embodiments, the one or more viral proteins encoded by the HIV-1 Gag gene do not comprise any p6 component. In some embodiments, the plurality of polypeptide fragments comprises at least 2 polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 68-146 and 339-342; SEQ ID NOs: 34-35; NO: 68, 69, 72, 73, 74, 75, 76, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 92, 93, 101, 102, 103, 104, 109, 110, 115, 116, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 139, 140, 141, 142, 143, 144, 145 and 146; SEQ ID NO: 76, 77, 86, 87 and 92-124; SEQ ID NO: 76, 77, 86, 87, 94 and 95; SEQ ID NO: 76, 86 and 94; SEQ ID NO: 77, 87 and 95; SEQ ID NO: 68-79 and 92-124; SEQ ID NO: 70-71, 76-77 and 94-95; SEQ ID NO: 78, 79, 96, 99, 100, 107, 108, 113, 114, 121, 122, 123, 124, 137 and 138; SEQ ID NO: 78, 99, 107, 113, 121, 123 and 137; SEQ ID NO: 78, 79, 90, 91, 97, 98, 99, 100, 105, 106, 107, 108, 111, 112, 113, 114, 117, 118, 119, 120, 121, 122, 123, 124, 137 and 138; NOs: 78, 90, 97, 105, 111, 117, 119 and 137; and SEQ ID NOs: 78 and 137.

[0391] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more fragments comprising or consisting of an HIV-1 Gag amino acid sequence corresponding to amino acid residue positions selected from the group consisting of 31-53, 37-51, 142-166, 175-199, 183-191, 257-282, 257-290, 265-282, 288-313, 288-321, 296-313, 333-357, 337-361, 341-349, 345-353, and 429-444 of SEQ ID NO: 1. In certain embodiments, the fusion polypeptide does not comprise one, two, three, four, five, or more polypeptide fragments comprising or consisting of an HIV-1 Gag amino acid sequence, or a subsequence thereof, corresponding to amino acid residue positions selected from the group consisting of 1-30, 54-127, 138-146, 370-428, and 445-500, wherein the amino acid positions are relative to SEQ ID NO: 404. In some embodiments, the plurality of polypeptide fragments does not include, or the fusion protein described herein does not include, 1, 2, 3, 4, 5, or more polypeptide fragments comprising or consisting of the HIV-1 Gag amino acid sequence of any one of SEQ ID NOs: 444-448, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 444-448, or a subsequence thereof. As used herein, when the position of any given polymer component (e.g., amino acid, nucleotide, also collectively referred to as "residue") is designated by reference to the same or equivalent position in a selected amino acid or nucleic acid polymer (e.g., based on an optimal alignment or consensus sequence), rather than by the actual numerical position of the component in the given polymer, the numbering of a given amino acid polymer or nucleic acid polymer "corresponds to," "corresponds to," or "is relative to" the numbering of a selected or reference amino acid polymer or nucleic acid polymer.

[0392] A polypeptide fragment encoded by the HIV-1 Nef gene

[0393] In some embodiments, the fusion polypeptide comprises one or more fragments of a viral protein encoded by the HIV-1 Nef gene. In some embodiments, the plurality of polypeptide fragments includes at least one polypeptide fragment, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more fragments comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, and 172; SEQ ID NOs: 147, 148, 149, 150, 155, 156, 157, 158, 159, 160, 166, 167, 168, 169, 170 and 171; SEQ ID NOs: 149-152; SEQ ID NOs: 151-152; SEQ ID NOs: 149, 150, 151, 152, 159, 160, 161, 162, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173 and 174; SEQ ID NOs: 151, 152, 161 and 162; SEQ ID NOs: 151 and 152; SEQ ID NOs: 153, 154, 172 and 173; SEQ ID NOs: 153 and 172; NOs: 153, 154, 155, 156, 157, 158, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, and 173; SEQ ID NOs: 153 and 165; and SEQ ID NO: 153.

[0394] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more fragments comprising or consisting of an HIV-1 Nef amino acid sequence corresponding to amino acid residue positions selected from the group consisting of 64-102, 81-102, 88-97, 91-99, 130-148, 130-154, 134-142, 134-148, 136-148, 137-145, 137-145, and 117-154, wherein the amino acid positions are relative to SEQ ID NO: 405. In certain embodiments, the fusion polypeptide does not include one, two, three, or more polypeptide fragments comprising or consisting of an HIV-1 Nef amino acid sequence, or a subsequence thereof, corresponding to amino acid residue positions selected from 1-63, 103-116, and 155-206, wherein the amino acid positions are relative to SEQ ID NO: 405. In some embodiments, the plurality of polypeptide fragments does not include, or the fusion protein described herein does not include, 1, 2, 3, or more polypeptide fragments comprising or consisting of the HIV-1 Nef amino acid sequence of any one of SEQ ID NOs: 449-451, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-451, or a subsequence thereof.

[0395] Fusion polypeptides having polypeptide fragments encoded by HIV-1 Gag and Nef genes

[0396] In some embodiments, the fusion polypeptide comprises or consists of one or more fragments of viral proteins encoded by the HIV-1 Gag and Nef genes, e.g., does not comprise one or more polypeptide fragments encoded by the HIV-1 Env, Pol, Tat, Rev, Vif, Vpr, or Vpu genes. In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: NOs: 68-79 and 92-124, 149, 150, 151, 152, 159, 160, 161, 162, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173 and 174; SEQ ID NOs: 70, 71, 76, 77, 94, 95, 151, 152, 161 and 162; SEQ ID NOs: 70, 76, 94, 151 and 161; and SEQ ID NOs: 71, 77, 95, 152 and 162.

[0397] In some embodiments, the fusion polypeptide comprises or consists of the following polypeptide fragments, optionally bound or linked by one or more linkers, in order from N-terminus to C-terminus: SEQ ID NOs: 70, 76, 94, 151, and 161; or SEQ ID NOs: 71, 77, 95, 152, and 162.

[0398] In some embodiments, the fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 351-356 and 430, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 351-356 and 430.

[0399] Modifications can be made in the structure of the fusion polypeptides described herein and the polynucleotides encoding such fusion polypeptides, and these modifications still result in functional molecules encoding variants or derivative polypeptides having desired (e.g., immunogenic) characteristics. When it is desired to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of a fusion polypeptide described herein, one skilled in the art will typically change one or more of the codons encoding the DNA sequence.

[0400] For example, certain amino acids can be substituted for other amino acids in a protein structure without significantly losing its ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding ability and properties of a protein that define the biological functional activity of that protein, certain amino acid sequence substitutions can be made in a protein sequence, and certainly in its underlying DNA coding sequence, while still obtaining a protein with similar properties. It is therefore contemplated that various changes can be made in the polypeptide sequences of the disclosed fusion polypeptides or the corresponding DNA sequences encoding such fusion polypeptides without significantly losing their biological utility or activity.

[0401] In many cases, polypeptide variants will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydrophilicity of the polypeptide would be substantially unchanged.

[0402] When comparing polynucleotide and polypeptide sequences, the two sequences are considered "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions (typically 30 to about 75, 40 to about 50) over which, after the two sequences have been optimally aligned, the sequence can be compared to a reference sequence having the same number of contiguous positions.

[0403] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W. J. Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0404] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm (Smith and Waterman (1981) Add. APL Math 2:482), the identity alignment algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443), the search similarity method (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444), computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or inspection.

[0405] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 methods, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine percent sequence identity for the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0406] In an illustrative example, for nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a wordlength (W) of 11 and an expectation (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) for alignments, (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains.

[0407] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0408] In one method, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less (usually 5% to 15%, or 10% to 12%) additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences (not comprising additions or deletions). The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0409] As used herein, the term polypeptide "variant" is a polypeptide that generally differs from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-described polypeptide sequences described herein and evaluating one or more biological activities of the polypeptides as described herein and / or using any of a variety of techniques well known in the art. The term "variant" may also refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0410] A polypeptide fragment encoded by the HIV-1 Env gene

[0411] In some embodiments, the fusion polypeptide comprises one or more fragments of one or more viral proteins encoded by the HIV-1 Env gene. In certain embodiments, the one or more viral proteins encoded by the HIV-1 Env gene are selected from gp120 and gp41.

[0412] In various embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-67 and 338; SEQ ID NOs: 1-67 and 338; NO: 2, 3, 8, 9, 13, 14, 17, 18, 23, 24, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 62, 63, 64, 65, 66 and 67; SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, SEQ ID NO: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 28, 29, 30, 37, 38, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 338; SEQ ID NO: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 28, 29, 30, 37, 38, 41, and 42; SEQ ID NO: 28, 29, 30, and 41-56; SEQ ID NO: 28, 29, 41, and 42; SEQ ID NO: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 37, and 38; NO: 4, 5, 11, 12, 37 and 38; SEQ ID NO: 6, 7, 15, 16, 21, 22, 30, 60 and 61; SEQ ID NO: 6, 15, 21, 30 and 60; SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 19, 20, 27, 55, 56, 57, 58, 59, 60, 61 and 338; SEQ ID NO: 1, 10, 19, 27, 55, 56 and 57; and SEQ ID NO: 6, 15 and 60.

[0413] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more fragments comprising or consisting of an HIV-1 Env amino acid sequence corresponding to amino acid residue positions selected from the group consisting of 28-52, 34-48, 34-47, 36-44, 59-83, 64-83, 66-83, 67-75, 113-137, 235-259, 586-594, 586-610, 589-606, and 594-602, wherein the amino acid positions are relative to SEQ ID NO: 403. In certain embodiments, the fusion polypeptide does not include 1, 2, 3, 4, 5, 6, or more polypeptide fragments comprising or consisting of an HIV-1 Env amino acid sequence or a subsequence thereof corresponding to amino acid residue positions selected from 1-27, 53-58, 84-112, 138-234, 269-474, 490-501, 611-856, wherein the amino acid positions are relative to SEQ ID NO: 403. In some embodiments, the plurality of polypeptide fragments does not include, or the fusion protein described herein does not include, 1, 2, 3, 4, 5, or more polypeptide fragments comprising or consisting of an HIV-1 Env amino acid sequence of any one of SEQ ID NOs: 437-443, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 437-443, or a subsequence thereof.

[0414] A polypeptide fragment encoded by the HIV-1 Pol gene

[0415] In some embodiments, the fusion polypeptide comprises one or more fragments of one or more viral proteins encoded by the HIV-1 Pol gene. In various embodiments, the one or more viral proteins encoded by the HIV-1 Pol gene are selected from one or more of protease (PR), reverse transcriptase (RT), and integrase (INT).

[0416] In some embodiments, the plurality of polypeptide fragments comprises at least 2 polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 174-337 and 343-344; SEQ ID NOs: 345-346; NO: 174, 175, 178, 179, 180, 181, 182, 183, 184, 185, 193, 194, 195, 196, 197, 198, 199, 200, 203, 204, 205, 206, 207, 208, 213, 214, 221, 222, 236, 237, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 266, 267, 268, 269, 270, 271, 272, 273, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 305, 306, 307, 308, 309, 310, 313, 314, 315, 316, 317, 318, 321 and 322; SEQ ID NOs: 180, 181, 182, 183, 184, 185, 186, 187, 190, 191, 192, 193, 194, 195, 196, 221, 222, 294, 295, 296, 297, 298, 299, 300, 301, 305, 306, 307, 308, 311, 312, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337; SEQ ID NOs: NO: 180, 181, 186, 187, 221, 222, 294, 295, 307, 308, 321 and 322;SEQ ID NO: 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 282, 283, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 and 337; SEQ ID NO: 176, 177, 188, 189, 213, 214, 223, 224, 259, 260, 282, 283, 294, 295, 305, 306, 319 and 320; SEQ ID NO: 180, 181, 186, 187, 221, 222, 294, 295, 321 and 322; SEQ ID NO: 182-202, 292-302, 305, 306; SEQ ID NO: 188, 189, 294, 295, 305, 306; SEQ ID NO: 176, 177, 178, 179, 180, 181, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, SEQ ID NOS: 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337;SEQ ID NOS: 192, 201, 202, 215, 216, 217, 218, 219, 220, 229, 230, 231, 240, 241, 242, 243, 244, 265, 276, 277, 298, 299, 302, 311, 312, 327, 328, 331, 332, 333, 336, and 337; SEQ ID NOS: 192, 201, 215, 217, 219, 229, 230, 240, 241, 243, 265, 276, 298, 302, 311, 327, 331, 333, and 336; NO: 190, 191, 192, 197, 198, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 21 9, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 239, 261, 262, 274, 275, 276, 277, 296, 297, 298, 299, 300, 301, 302, 303, 304, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 343, 344, 375 and 376; SEQ ID NO: 190, 197, 209, 210, 211, 225, 227, 234, 238, 261, 296, 300, 303, 323, 325, 329, 334; and SEQ ID NO: 192, 215, 217, 219, 229, 230, 276, 298, 302, 327, 331, 333, and 336.

[0417] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30 or more of the following fragments: These fragments contain a polypeptide fragment selected from 144-168, 152-160, 291-315, 326-350, 328-352, 330-354, 333-354, 334-342, 336-344, 337-355, 338-356, 339-361, 340-341, 341-363, 342-343, 344-345, 346-347, 348-349, 350-351, 351-352, 352-353, 353-354, 354-356, 356-357, 357-358, 358-359, 359-360, 361-361, 362-363, 363-364, 364-365, 365-366, 367-368, 368-369, 369 ... 964, 980-1003, and 988-996, wherein the amino acid positions are relative to SEQ ID NO: 406. In certain embodiments, the fusion polypeptide does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptide fragments comprising or consisting of an HIV-1 Pol amino acid sequence corresponding to amino acid residue positions selected from 1-55, 118-128, 321-325, 355-366, 432-541, 607-641, 667-682, 709-746, 828-833, 921-930, or a subsequence thereof, wherein the amino acid positions are relative to SEQ ID NO: 406. In some embodiments, the plurality of polypeptide fragments does not include, or the fusion protein described herein does not include, 1, 2, 3, 4, 5, or more polypeptide fragments comprising or consisting of an HIV-1 Pol amino acid sequence of any one of SEQ ID NOs: 452-461, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 452-461, or a subsequence thereof.

[0418] In some embodiments, the fusion polypeptide comprising a polypeptide fragment encoded by the HIV-1 Pol gene does not comprise the amino acid sequence or motif YMDD (SEQ ID NO: 462) or YVDD (SEQ ID NO: 463). In some embodiments, the fusion polypeptide does not comprise one or more amino acid sequences selected from SEQ ID NOs: 215, 216, 217, 218, 219, and 220. In some embodiments, the fusion polypeptide does not comprise one or more amino acid sequences selected from SEQ ID NOs: 209, 210, 211, 212, 213, 214, 343, and 344.

[0419] Fusion polypeptides having polypeptide fragments encoded by HIV-1 Env and Pol genes

[0420] In some embodiments, the fusion polypeptide comprises or consists of one or more fragments of viral proteins encoded by the HIV-1 Env and Pol genes, e.g., does not comprise one or more polypeptide fragments encoded by the HIV-1 Gag, Nef, Tat, Rev, Vif, Vpr, or Vpu genes.

[0421] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: NO: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 28, 29, 30, 37, 38, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224 4, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 282, 283, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 and 338; SEQ ID NOs: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 28, 29, 30, 37, 38, 41, 42, 176, 177, 188, 189, 213, 214, 223, 224, 259, 260, 282, 283, 294, 295, 305, 306, 319, and 320; SEQ ID NOs: 28, 29, 30, 41-56, 182-202, 292-302, 305, and 306; SEQ ID NOs: 28, 29, 41, 42, 188, 189, 294, 295, 305, and 306;SEQ ID NO: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 37, 38, 176, 177, 178, 179, 180, 181, 213, 214, 215, 216, 217, 2 18, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 2 and SEQ ID NOs: 4, 5, 11, 12, 37, 38, 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337; and SEQ ID NOs: 4, 5, 11, 12, 37, 38, 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319, and 320.

[0422] In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, the following polypeptide fragments, optionally bound or connected by one or more linkers: SEQ ID NOs: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213, and 37; SEQ ID NOs: 188, 305, 28, 41, and 294; SEQ ID NOs: 4, 176, 11, 319, 259, 282, 223, 213, and 37; SEQ ID NOs: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214, and 38; SEQ ID NO: 5, 177, 12, 320, 260, 283, 224, 214 and 38; SEQ ID NO: 305, 319, 259, 282, 223, 213, 294, 176 and 188; SEQ ID NO: 306, 320, 260, 283, 224, 214, 295, 177 and 189; SEQ ID NO: 305, 294, 223, 213, 176, 259, 319, 188 and 282; SEQ ID NO: 306, 295, 224, 214, 177, 260, 320, 189 and 283; SEQ ID NO: 305, 294, 319, 259, 282, 223, 176 and 188; NO: 306, 295, 320, 260, 283, 224, 177 and 189; SEQ ID NO: 305, 223, 294, 176, 259, 319, 188 and 282; or SEQ ID NO: 306, 224, 295, 177, 260, 320, 189 and 283.

[0423] In some embodiments, the fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 357-366 and 407-410, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 357-366 and 407-410, wherein the percent identity is across the entire length of SEQ ID NOs: 357-366 and 407-410.

[0424] Fusion polypeptides containing polypeptide fragments encoded by HIV-1 Gag, Nef, and Pol genes

[0425] In some embodiments, the fusion polypeptide comprises or consists of one or more fragments of viral proteins encoded by the HIV-1 Gag, Nef, and Pol genes, e.g., does not comprise one or more polypeptide fragments encoded by the HIV-1 Env, Tat, Rev, Vif, Vpr, or Vpu genes.

[0426] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: NO: 76, 77, 86, 87, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 1 13, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 180, 181, 182, 183, 184, 185, 186, 1 87, 190, 191, 192, 193, 194, 195, 196, 221, 222, 294, 295, 296, 297, 298, 299, 300, 301, 305, 306, 307, 308, 311, 312, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 340, 341 and 342; and SEQ. ID NO: 76, 77, 86, 87, 94, 95, 151, 152, 181, 182, 186, 187, 221, 222, 294, 195, 307, 308, 321, 322.

[0427] In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, the following polypeptide fragments: SEQ ID NO: 76, 86, 94, 180, 186, 221, 294, 307, 321, and 151; or SEQ ID NO: 77, 87, 95, 181, 187, 222, 295, 308, 322, and 152, optionally bound or connected by one or more linkers.

[0428] In some embodiments, the fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 345-350 and 422-424, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 345-350 and 422-424, wherein the percent identity is across the entire length of SEQ ID NOs: 345-350 and 422-424.

[0429] In some embodiments, the fusion protein comprises or consists of an amino acid sequence that is any one of SEQ ID NOs: 527-528. In some embodiments, the fusion polypeptide comprises or consists of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of SEQ ID NOs: 527-528.

[0430] It has the following components: HIV-1 Env, Gag, Nef and Pol and is predicted to bind to or be bound by the human HLA A*0201 molecule Fusion polypeptides of peptide fragments presented by molecules

[0431] As described herein, we have designed fusion polypeptides comprising multiple polypeptide segments that are computationally predicted to bind to or be presented by human HLA A*0201 molecules. Typically, the polypeptide segments selected or included in such fusion polypeptides comprise polypeptide segments encoded by the HIV-1 Env, Gag, Nef, and Pol genes (e.g., do not include one or more polypeptide segments encoded by the HIV-1 Tat, Rev, Vif, Vpr, or Vpu genes), and are predicted to bind to human HLA A*0201 molecules with an IC50 value of less than about 1,000 nM. In certain embodiments, the polypeptide segments selected or included in such fusion polypeptides comprise polypeptide segments encoded by the HIV-1 Env, Gag, Nef, and Pol genes (e.g., do not include one or more polypeptide segments encoded by the HIV-1 Tat, Rev, Vif, Vpr, or Vpu genes), and are predicted to bind to human HLA A*0201 molecules at a percentile rank within the top 5% of the population of polypeptide segments. In some embodiments, the fusion polypeptide comprises or consists of fragments of viral proteins encoded by the Gag, Pol, Env, and Nef genes, wherein each of the multiple polypeptide fragments can bind to or be presented by the human HLA allele A*0201.

[0432] In some embodiments, each of the plurality of polypeptide fragments is 8 to 35 amino acids in length, eg, 9 to 34 amino acids in length, eg, 9 to 25 amino acids in length.

[0433] In some embodiments, the fusion polypeptide comprises at least two polypeptide fragments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more of the following fragments: the fragments comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: SEQ ID NOS: 6, 7, 15, 16, 21, 22, 30, 60, 61, 78, 79, 96, 99, 100, 107, 108, 113, 114, 121, 122, 123, 124, 137, 138, 153, 154, 172, 173, 192, 201, 202, 215, 216, 217, 218, 219, 220, 229, 230, 231, 240, 241, 242, 243, 244, 265, 276, 277, 298, 299, 302, 311, 312, 327, 328, 331, 332, 333, 336, and 337; NO: 6, 15, 21, 30, 60, 78, 99, 107, 113, 121, 123, 137, 153, 172, 192, 201, 215, 217, 219, 229, 230, 240, 241, 243, 265, 276, 298, 302, 311, 327, 331, 333 and 336;SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 19, 20, 27, 55, 56, 57, 58, 59 ,60,61,78,79,90,91,97,98,99,100,105,106,107,108,111,112,113,1 14, 117, 118, 119, 120, 121, 122, 123, 124, 137, 138, 153, 154, 155, 156, 15 7, 158, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 190, 191, 192 , 197, 198, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 239, 261, 262, 274, 275, 276, 277, 296, 297, 298, 299, 300, 301, 302, 303, 304, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 343 and 344; ID NO: 1, 10, 19, 27, 55, 56, 57, 78, 90, 97, 105, 111, 117, 119, 137, 153, 165, 190, 197, 209, 210, 211, 225, 227, 234, 238, 261, 296, 300, 303, 323, 325, 329 and 334.

[0434] In some embodiments, the fusion polypeptide comprises, in order from N-terminus to C-terminus, the following polypeptide fragments, optionally bound or connected by one or more linkers: SEQ ID NO: 201, 78, 107, 96, 229, 172, 327, 6, 333, 243, 331, 192, 265, 311, 137, 15, 123, 30, 336, 302, 153, 219, 298, 121, 230, 240, 60, 241, 276, 113, 99, 21, 217, and 215; NO: 78, 296, 1, 339, 197, 329, 232, 323, 303, 234, 90, 261, 274, 238, 211, 325, 137, 227, 209, 190, 341, 57, 225, 27, 210, 119, 19, 165, 334, 117, 153, 10, 97, and 300; or SEQ ID NO: 296, 1, 78, 197, 339, 227, 261, 274, 238, 325, 137, 329, 303, 234, 90, 232, 27, 57, 225, 323, 190, 341, 119, 19, 165, 334, 117, 153, 10, 97 and 300.

[0435] In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 367-377 and 411, or a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 367-377 and 411, wherein the percent identity is across the entire length of SEQ ID NOs: 367-377 and 411.

[0436] Signal or leader sequence

[0437] In various embodiments, the fusion polypeptide includes a signal sequence or signal peptide, for example, to guide the fusion polypeptide to be transported to the proteasome or lysosomal compartment within the cell. In various embodiments, the fusion polypeptide includes a signal sequence at the N-terminus and / or the C-terminus. In some embodiments, the fusion polypeptide includes an N-terminal signal peptide or leader sequence. In various embodiments, the signal peptide or leader sequence is from a source protein selected from serum proteins, cytokines, chemokines, chaperones, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In some embodiments, the signal peptide or leader sequence is from a source protein selected from the group consisting of colony stimulating factor 2 (CSF2, GM-CSF), tissue plasminogen activator (PLAT, t-PA), CC motif chemokine ligand 7 (CCL7, MCP-3), CXC motif chemokine ligand 10 (CXCL10, IP-10), catenin beta 1 (CTNNB1), CD74 (p33; DHLAG; HLADG; Ia-GAMMA, invariant chain), serum albumin (ALB), polyubiquitin B / C (UBB / UBC), calreticulin (CALR), vesicular stomatitis virus G protein (VSV-G), lysosomal associated membrane protein 1 (LAMP-1), and lysosomal associated membrane protein 2 (LAMP-2). In certain embodiments, the fusion polypeptide comprises the N-terminal and C-terminal signal sequences from LAMP-1, e.g., SEQ ID NOs: 399 and 412, respectively. In various embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs: 393-402 and 412-413, or a sequence at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 393-402 and 412-413, wherein the percent identity is across the entire length of SEQ ID NOs: 393-402 and 412-413.

[0438] Polynucleotide encoding fusion polypeptide

[0439] Provided are polynucleotides encoding fusion polypeptides described herein (e.g., HIV immunogenic polypeptides), expression cassettes, expression vectors, and viral vectors comprising such polynucleotides, and host cells (e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, bacterial cells, such as E. coli) comprising such polynucleotides, expression cassettes, expression vectors, or viral vectors. As used herein, the phrase "HIV immunogenic nucleic acid sequence" refers to a polynucleotide encoding an HIV immunogenic polypeptide (e.g., a fusion polypeptide).

[0440] Provided herein are polynucleotides comprising the nucleotide sequence of any one of the fusion polypeptides provided herein for encoding, and expression cassettes, expression vectors, and viral vectors comprising such polynucleotide sequences, for example, for expression vectors effectively expressed in host cells (e.g., mammalian cells). In various embodiments, the polynucleotides are DNA, cDNA, mRNA, self-amplification RNA (SAM), self-replicating RNA, or self-amplification replicon RNA (RepRNA). In some embodiments, the polynucleotides include alphavirus self-replication or self-amplification replicon RNA (RepRNA). Self-replicating RNA and self-amplifying replicon RNA as vaccine delivery modes are described, for example, in Tews et al., Methods Mol Biol. (2017) 1499: 15-35; Démoulins et al., Methods Mol Biol. (2017) 1499: 37-75; Englezou et al., Mol Ther Nucleic Acids. (2018) 12: 118-134; McCollough et al., Vaccines (Basel). (2014) 2(4): 735-54; and McCollough et al., Mol Ther Nucleic Acids. (2014) 3: e173.

[0441] The terms "polynucleotide" and "nucleic acid molecule" refer interchangeably to polymeric forms of nucleotides, and include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers thereof. As used herein, the term "nucleic acid molecule" is interchangeable with the term "polynucleotide." In some embodiments, nucleotides refer to ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide, such as a cDNA or mRNA, may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), side chain moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelating agents, alkylates, and modified bonds (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded conformation, double-stranded conformation, partially double-stranded conformation, triple-stranded conformation, hairpin conformation, circular conformation, and padlock conformation. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complementary sequence. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain and its complementary sequence. The term also includes codon-biased polynucleotides for improving expression in desired viral expression vectors or host cells.

[0442] As used herein, "substitution" means that one or more amino acids or nucleotides are replaced by different amino acids or nucleotides, respectively.

[0443] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated nucleic acid encoding a polypeptide fragment or encoding a fusion polypeptide" refers to one or more nucleic acid molecules encoding such a polypeptide fragment or fusion polypeptide, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

[0444] As used herein, the term polynucleotide "variant" is a polynucleotide that typically differs from the polynucleotides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the polynucleotide sequences described herein and evaluating one or more biological activities of the encoded polypeptides as described herein and / or using any of a variety of techniques well known in the art.

[0445] In some embodiments, the nucleic acid molecule has a codon preference to enhance expression in a desired host cell (e.g., a human cell, a mammalian cell, a yeast cell, a plant cell, an insect cell, or a bacterial cell, such as an E. coli cell). Thus, polynucleotides encoding the fusion polypeptides described herein are provided, wherein the polynucleotide has a codon preference, comprises a replacement heterologous signal sequence, and / or eliminates an mRNA instability element. Methods for generating codon-biased nucleic acids can be performed by adapting the methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. Preferred codon usage for expressing a fusion polypeptide comprising an HIV-1 polypeptide fragment from a desired viral expression vector and / or in a desired host cell is provided, for example, at kazusa.or.jp / codon / and genscript.com / tools / codon-frequency-table.

[0446] In some embodiments, the polynucleotide encoding the fusion polypeptide as described herein has a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 414-418, wherein the percent identity spans the entire length of SEQ ID NOs: 414-418.

[0447] In some embodiments, the polynucleotide encoding the fusion polypeptide as described herein has a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 524-526, wherein the percent identity spans the entire length of SEQ ID NOs: 524-526.

[0448] In some embodiments, the one or more polynucleotides encode one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435, or encode one or more fusion proteins comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435.

[0449] In various embodiments, the two or more polynucleotides encode two or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 350 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 369 and 370, SEQ ID NOs: 371 and 372, SEQ ID NOs: 373 and 374, SEQ ID NOs: 375 and 376, SEQ ID NOs: 377 and 378, SEQ ID NOs: 379 and 380, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 431, SEQ ID NOs: 431 and 432, SEQ ID NOs: 432 and 433, SEQ ID NOs: 433 and 434. NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410; or SEQ ID NOs: 409 and 410.

[0450] In various embodiments, the polynucleotide encodes a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that has 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that has 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that has 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein with 4 or fewer amino acid mismatches to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein with 3 or fewer amino acid mismatches to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein with 2 or fewer amino acid mismatches to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein with 1 or fewer amino acid mismatches to the amino acid sequence of SEQ ID NO: 527.

[0451] In various embodiments, the polynucleotide encodes a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 4 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 3 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 2 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that has 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528.

[0452] In various embodiments, the one or more polynucleotides encode one or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, and 430-435.

[0453] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 524.

[0454] In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0455] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0456] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the polynucleotide comprises the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0457] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0458] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0459] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0460] In various embodiments, the polynucleotide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 15 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide has 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the polynucleotide comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 521.

[0461] Optionally, in certain embodiments, the 3' end of a polynucleotide encoding a fusion polypeptide described herein comprises one or more tandem stop codons, for example, two or more tandem TAG ("amber"), TAA ("ochre"), or TGA ("cream" or "umber") stop codons. The multiple tandem stop codons may be the same or different.

[0462] Also provided is an expression cassette comprising a polynucleotide encoding a fusion polypeptide as described herein, the polynucleotide being operably linked to one or more regulatory sequences. In some embodiments, the polynucleotide is operably linked to and under the control of a constitutive promoter. In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus major immediate early promoter (CMV), a CMV enhancer fused to a chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1ɑ), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK).

[0463] Expression cassettes, vectors, viral vectors and host cells

[0464] Also provided are vectors comprising one or more polynucleotides encoding one or more fusion polypeptides described herein, or or expression cassettes comprising such polynucleotides. The vector can be of any type, such as a recombinant vector, such as an expression vector. The vector includes, but is not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) and vectors derived from bacteriophages or plants or animals (including humans) viruses. The vector can include a replication origin recognized by the proposed host cell, and with respect to expression vectors, includes a promoter and other regulatory regions recognized by the host cell. In other embodiments, the vector includes one or more polynucleotides encoding one or more fusion polypeptides of the present disclosure, which are operably connected to a promoter and optionally other regulatory elements. Some vectors can replicate autonomously in the host into which they are introduced (for example, vectors with a bacterial replication origin can replicate in bacteria). Other vectors can be integrated into the host's genome when introduced into the host, thereby replicating together with the host genome. The vector includes, but is not limited to, those suitable for recombinant production of fusion polypeptides disclosed herein.

[0465] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which the vector has been introduced. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as expression vectors.

[0466] The term "operably linked" refers to two or more nucleic acid sequence elements that are typically physically linked and in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control of the promoter."

[0467] The selection of vector depends on the subsequent recombination program and the host used. The introduction of vector into the host cell can be achieved by especially calcium phosphate transfection, DEAE-dextran mediated transfection, lipofectamine transfection, electroporation, viral infection or via administration to the subject as described herein. The vector can replicate autonomously or can replicate together with the chromosome into which it is integrated. In certain embodiments, the vector contains one or more selection markers. The selection of markers can depend on the host cell selected. These include but are not limited to kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene (HSV-TK) from herpes simplex virus and dihydrofolate reductase gene (dhfr) from mouse. The present disclosure also encompasses vectors comprising one or more nucleic acid molecules encoding the fusion polypeptide as described herein, which are operably linked to one or more nucleic acid molecules encoding a protein or peptide ("purification tag") that can be used to separate the fusion polypeptide. These proteins or peptides include, but are not limited to, FLAG tag (DYKDDDDKL; SEQ ID NO: 436), glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and β-galactosidase.

[0468] In other embodiments, the vector used is pcDNA TM 3.1+ (ThermoFisher, MA).

[0469] In some embodiments, the vector is a viral expression vector or a viral vector. As used herein, a viral expression vector refers to a viral expression system or a viral expression construct, and a viral vector refers to a viral particle. Depending on the circumstances, the viral expression vector or viral vector can be a DNA virus or an RNA virus, including a self-replicating RNA virus. Self-replicating RNA viruses include alphaviruses and are described, for example, in Lundstrom, Molecules. (2018) 23 (12). pii: E3310 (PMID: 30551668); and Ljungberg et al., Expert Rev Vaccines. (2015) 14 (2): 177-94). In various embodiments, the viral expression vector or viral vector is from a virus selected from the group consisting of adenovirus, adeno-associated virus, arenavirus, alphavirus, self-replicating alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus, and vaccinia virus. In some embodiments, the viral expression vector or viral vector is from a virus family selected from the group consisting of: Adenoviridae (e.g., adenovirus, adeno-associated virus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus, Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus)), Herpesviridae (e.g., cytomegalovirus, herpesvirus, such as HSV-1), Parvoviridae (e.g., Parvovirus H1), Poxviridae (e.g., vaccinia virus, such as Modified Vaccinia Ankara virus (MVA)). , Paramyxoviridae (e.g., measles virus), Flaviviridae (e.g., yellow fever virus), Reoviridae (e.g., reovirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesiculoviruses, including Maraba vesiculovirus and vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphaviruses, e.g., self-replicating alphaviruses; Sindbis virus), Enteroviridae (e.g., echoviruses). Exemplary modified vaccinia virus expression vectors or viral vectors for expressing the fusion polypeptides of the present invention are described, for example, in WO 2019 / 134049.

[0470] In some embodiments, the viral expression vector or viral vector is an arenavirus vector selected from the group consisting of: lymphocytic choriomeningitis mammalian arenavirus (LCMV) (NCBI: txid11623), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus) (NCBI: txid2169993), Guanarito virus (GTOV) (NCBI: txid45219), Argentinian mammalian arenavirus (also known as Junin virus (JUNV) ))(NCBI:txid2169991), Lassa virus (LASV) (NCBI:txid11620), Luyo virus (LUJV) (NCBI:txid649188), Machupo virus (MACV) (NCBI:txid11628), Brazilian mammalian arenavirus (also known as Sabia virus (SABV)) (NCBI:txid2169992), and Whitewater Arroyo virus (WWAV) (NCBI:txid46919). In some embodiments, the viral expression vector or viral vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus). Exemplary arenavirus vectors that can be used as delivery and expression vehicles for the fusion polypeptides described herein are described, for example, in WO 2009 / 083210, WO 2015 / 183895, WO 2016 / 075250, WO 2017 / 198726, and U.S. Pat. No. 9,943,585.

[0471] In some embodiments, the viral expression vector or viral vector is an adenovirus vector, e.g., from a human adenovirus or a simian adenovirus (e.g., a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus macaque adenovirus). In some embodiments, the adenovirus vector is selected from serotype 5 adenovirus (Ad5), serotype 26 adenovirus (Ad26), serotype 34 adenovirus (Ad34), serotype 35 adenovirus (Ad35), serotype 48 adenovirus (Ad48), chimpanzee adenovirus (e.g., ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (Ad11), ChAd12 (Ad13), ChAd13 (Ad14), ChAd14 (Ad15), ChAd15 (Ad16), ChAd17 (Ad18), ChAd19 (Ad19), ChAd20 (Ad21), ChAd21 (Ad22), ChAd22 (Ad23), ChAd23 (Ad24), ChAd24 (Ad25), ChAd25 (Ad26), ChAd27 (Ad27), ChAd28 (Ad29), ChAd29 (Ad30), ChAd30 (Ad31), ChAd31 (Ad32), ChAd32 (Ad33), ChAd33 (Ad34), ChAd33 (Ad35), ChAd34 (Ad35), ChAd35 (Ad36), ChAd36 (Ad37), ChAd37 (Ad38), ChAd38 (Ad39), ChAd39 (Ad31), ChAd3 (AdC10), ChAd11(AdC11), ChAd17(AdC17), ChAd16(AdC16), ChAd19(AdC19), ChAd20(AdC20), ChAd22(AdC 22), ChAd24(AdC24), ChAdY25, ChAd26(AdC26), ChAd28(AdC28), ChAd30(AdC30), ChAd31(AdC31), ChAd37( AdC37), ChAd38(AdC38), ChAd43(AdC43), ChAd44(AdC44), ChAd55(AdC55), ChAd63(AdC63), ChAdV63, ChA d68(AdC68), ChAd73(AdC73), ChAd82(AdC82), ChAd83(AdC83), ChAd143(AdC143), ChAd144(AdC144), ChAd 145 (AdC145), ChAd147 (AdC147)), gorilla adenovirus (e.g., GC44, GC45, GC46), and rhesus macaque adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66).Exemplary chimpanzee, gorilla, and rhesus monkey adenoviral vectors that can be used as delivery and expression vehicles for the fusion polypeptides described herein are described, for example, in WO 2019 / 076880; WO 2019 / 076877; Andrabi et al., (2019) Cell Reports 27:2426-2441; Guo et al., Hum Vaccin Immunother. (2018) 14(7):1679-1685; Abbink et al., J Virol. (2015) 89(3):1512-22; and Abbink et al., J Virol. (2018) 92(6).pii:e01924-17.

[0472] In various embodiments, the viral expression vector or viral vector is unable to replicate (ie, replication-defective or replication-deficient), eg, has reduced or attenuated replication ability compared to a wild-type viral vector (ie, replication-attenuated), or is replication-competent.

[0473] In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector is an adenoviral expression vector or viral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435, or encoding one or more fusion proteins comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435.

[0474] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises two or more polynucleotides encoding two or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the following amino acid sequence: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 350 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 369 and 370, SEQ ID NOs: 371 and 372, SEQ ID NOs: 373 and 374, SEQ ID NOs: 375 and 376, SEQ ID NOs: 377 and 378, SEQ ID NOs: 379 and 380, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 431, SEQ ID NOs: 431 and 432, NO: 430 and 359, SEQ ID NO: 430 and 360, SEQ ID NO: 430 and 361, SEQ ID NO: 430 and 362, SEQ ID NO: 407 and 409, SEQ ID NO: 407 and 408, SEQ ID NO: 408 and 410; or SEQ ID NO: 409 and 410.

[0475] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527.In some embodiments, an expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, an expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 4 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, an expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 3 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, an expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 2 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, an expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527.

[0476] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 4 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 3 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 2 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide encoding a fusion protein having 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528.

[0477] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises one or more polynucleotides encoding one or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, and 430-435.

[0478] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:524.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0479] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:525.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0480] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:526.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0481] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:522.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0482] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO:523.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0483] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:520.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0484] In various embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO:521.In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the expression cassette, expression vector, viral expression vector, or viral vector comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 521.

[0485] Figure 14A A linear schematic diagram of a viral vector comprising a promoter sequence, an immunogen sequence (e.g., a sequence encoding an immunogenic polypeptide (such as an HIV immunogenic polypeptide or fusion polypeptide)), and a polyA sequence is shown. Exemplary viral vector sequences, such as SEQ ID NOs: 520, 521, 534, and 535, and the nucleotide sequences corresponding to the promoter, immunogen, and polyA sequences are also shown. Figure 14A In some embodiments, the CMV promoter sequence corresponding to nucleotides 594 to 1195 of SEQ ID NOs: 520, 521, 534, and 535 is replaced with another promoter sequence. In other words, in some embodiments, the CMV promoter of any of the viral vectors disclosed herein (such as SEQ ID NOs: 520, 521, 534, or 535) is replaced with any of the promoters disclosed herein (such as the SV40 promoter). In some embodiments, the immunogenic sequence corresponding to nucleotides 1256 to 4351 of SEQ ID NO: 520, or nucleotides 1256 to 4348 of SEQ ID NO: 521, or nucleotides 1256 to 2779 of SEQ ID NO: 534, or nucleotides 1256 to 3358 of SEQ ID NO: 535 is replaced with any of the immunogenic sequences disclosed herein. In some embodiments, the immunogenic sequence disclosed herein constitutes any of the HIV immunogenic polypeptides or fusion proteins disclosed herein. In some embodiments, the polyA corresponding to nucleotides 4596 to 4619 of SEQ ID NO: 520, or nucleotides 4393 to 4616 of SEQ ID NO: 521, or nucleotides 2824 to 3047 of SEQ ID NO: 534, or nucleotides 3406 to 3629 of SEQ ID NO: 535 is replaced with another polyA sequence.

[0486] In some embodiments, the viral vector SEQ ID NO: 520 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 4351 of SEQ ID NO: 520 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 525 or 526, wherein the percent identity is across the entire length of SEQ ID NO: 525 or 526. In some embodiments, the viral vector SEQ ID NO: 520 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 4351 of SEQ ID NO: 520 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0487] In some embodiments, the viral vector SEQ ID NO:521 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 4348 of SEQ ID NO:521 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:524, wherein the percent identity is across the entire length of SEQ ID NO:524. In some embodiments, the viral vector SEQ ID NO: 521 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 4348 of SEQ ID NO: 521 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0488] In some embodiments, the viral vector SEQ ID NO: 534 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 2779 of SEQ ID NO: 534 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 524, 525 or 526, wherein the percent identity is across the entire length of SEQ ID NO: 524, 525 or 526. In some embodiments, the viral vector SEQ ID NO: 534 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 2779 of SEQ ID NO: 534 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0489] In some embodiments, the viral vector SEQ ID NO: 535 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 3358 of SEQ ID NO: 535 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 524, 525 or 526, wherein the percent identity is across the entire length of SEQ ID NO: 524, 525 or 526. In some embodiments, the viral vector SEQ ID NO: 535 is modified such that the immunogenic sequence corresponding to nucleotides 1256 to 3358 of SEQ ID NO: 535 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0490] Figure 14BA linear schematic diagram of a viral vector comprising a promoter sequence, an immunogen sequence (e.g., a sequence encoding an immunogenic polypeptide (such as an HIV immunogenic polypeptide or fusion polypeptide)), and a polyA sequence is shown. Exemplary viral vector sequences, such as SEQ ID NOs: 522, 523, and 537, and the nucleotide sequences corresponding to the promoter, immunogen, and polyA sequences are also shown. Figure 14B In some embodiments, the 26S subgenomic promoter sequence corresponding to nucleotides 7513 to 7536 of SEQ ID NOs: 522, 523, and 537 is replaced with another subgenomic promoter sequence. In other words, in some embodiments, the 26S subgenomic promoter of any of the viral vectors disclosed herein (such as SEQ ID NOs: 522, 523, and 537) is replaced with any of the promoters disclosed herein (such as a subgenomic promoter derived from an alphavirus). In some embodiments, the immunogenic sequence corresponding to nucleotides 7571 to 10666 of SEQ ID NO: 522, or nucleotides 7571 to 10663 of SEQ ID NO: 523, or nucleotides 7571 to 9673 of SEQ ID NO: 538 is replaced with any of the immunogenic sequences disclosed herein. In some embodiments, the immunogenic sequence disclosed herein constitutes any of the HIV immunogenic polypeptides or fusion proteins disclosed herein. In some embodiments, the polyA corresponding to nucleotides 10953 to 11072 of SEQ ID NO: 522, or nucleotides 10950 to 11069 of SEQ ID NO: 523, or nucleotides 9960 to 10039 of SEQ ID NO: 537 is replaced with another polyA sequence.

[0491] In some embodiments, the viral vector SEQ ID NO: 522 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 10666 of SEQ ID NO: 522 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 525 or 526, wherein the percent identity is across the entire length of SEQ ID NO: 525 or 526. In some embodiments, the viral vector SEQ ID NO: 522 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 10666 of SEQ ID NO: 522 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0492] In some embodiments, the viral vector SEQ ID NO:523 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 10663 of SEQ ID NO:523 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO:524, wherein the percent identity is across the entire length of SEQ ID NO:524. In some embodiments, the viral vector SEQ ID NO: 523 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 10663 of SEQ ID NO: 523 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0493] In some embodiments, the viral vector SEQ ID NO: 537 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 9673 of SEQ ID NO: 537 is replaced with a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the nucleotide sequence of SEQ ID NO: 524, 525 or 526, wherein the percent identity is across the entire length of SEQ ID NO: 524, 525 or 526. In some embodiments, the viral vector SEQ ID NO: 537 is modified such that the immunogenic sequence corresponding to nucleotides 7571 to 9673 of SEQ ID NO: 537 is replaced with a nucleotide sequence that encodes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is across the entire length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0494] In some embodiments, the expression vector, viral expression vector or viral vector further comprises a polynucleotide encoding a cytokine or a functional variant thereof, or a non-coding immunostimulatory polynucleotide. In some embodiments, the expression vector, viral expression vector or viral vector further comprises a polynucleotide encoding a cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, colony stimulating factor 2 (CSF2; also known as GM-CSF), fms-related receptor tyrosine kinase 3 ligand (FLT3LG), and combinations and functional variants thereof. Co-expression and / or co-administration of cytokines with vaccines are described, for example, in Elizaga et al. (2018) PLoS One 13(9):e0202753 (IL-12); Buchbinder et al. (2017) PLoS One 12(7):e0179597 (GM-CSF); Abaitua et al., Virus Res (2006) 116(1-2):11-20 (IL12+IFN-γ); Oudard et al., Cancer Immunol Immunother (2011) February;60(2):261-71 (IL-2+IFN-α). In some embodiments, the vector (e.g., an expression vector, a viral expression vector, or a viral vector) further comprises a non-coding immunostimulatory polynucleotide selected from a pathogen-activated molecular pattern (PAMP), a cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide, and an immunostimulatory RNA (isRNA). Exemplary isRNAs include CV8102 (CureVac) and others described, e.g., in WO2016170176.

[0495] Also provided are host cells comprising one or more polynucleotides encoding one or more fusion polypeptides described herein or one or more vectors expressing the fusion polypeptides. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as Escherichia coli. In another embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, a mammalian cell, such as a cell line based on Chinese hamster ovary (CHO) or CHO-derived (e.g., CHO-S, CHO DG44, ExpiCHO, or similar). TM 、 ZFN-modified GS- / - CHO cell lines, CHO-K1, CHO-K1a), COS cells, BHK cells, NSO cells or Bowes melanoma cells. Examples of human host cells include HeLa, 911, AT1080, A549 and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293 TM Additionally, the fusion polypeptide can be expressed in yeast cells such as Pichia pastoris (see, eg, Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hanseula, or Saccharomyces.

[0496] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0497] As the case may be, the host cell can be stably or transiently transfected with one or more polynucleotides encoding one or more fusion polypeptides as described herein. As the case may be, the host cell can be infected with one or more vectors expressing one or more fusion polypeptides as described herein. In some embodiments, the host cell is capable of expressing one or more replication-attenuated or replication-competent vectors of one or more fusion polypeptides as described herein and is infected or capable of propagating the vector. Exemplary cells suitable for being infected and / or propagating viral vectors include, but are not limited to, BHK-21, A549, Vero, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293 TM ) cells. In certain embodiments, the host cell expresses a coxsackievirus and adenovirus receptor (CAR), such as MDCK, Caco-2, or Calu-3 host cells. In certain embodiments, the polynucleotide is integrated into the genome of the host cell.

[0498] Lipid nanoparticles, polymer nanoparticles, and nanoemulsions

[0499] In certain embodiments, any of the polynucleotides or vectors disclosed herein are formulated into lipid nanoparticles. For example, in some embodiments where the fusion polypeptide is expressed from a self-replicating or self-amplifying RNA molecule, the self-replicating or self-amplifying RNA can be formulated into lipoplexes, such as lipid nanoparticles (LNPs), polymeric nanoparticles (PNPs), and nanoemulsions. As used herein, "lipoplexes" refer to cationic liposomes that are non-viral (synthetic) lipid carriers for DNA.

[0500] As used herein, the term "lipid nanoparticle" refers to one or more spherical nanoparticles having an average diameter between about 10 nanometers and about 1000 nanometers and comprising a solid lipid core matrix that can dissolve lipophilic molecules. In certain embodiments, the lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and can include one or more of the following: triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glyceryl monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Pat. Nos. 6,217,912, 6,881,421, 7,402,573, 7,404,969, 7,550,441, 7,727,969, 8,003,621, 8,691,750, 8,871,509, 9,017,726, 9,173,853, 9,220,779, 9,227,917, and 9,278,130, each of which is incorporated by reference in its entirety. In one embodiment, the self-replicating or self-amplifying RNA molecules encoding one or more fusion polypeptides described herein are formulated or condensed into a polyethyleneimine (PEI)-complex delivery vehicle, such as described in Démoulins et al., Nanomedicine. (2016) April; 12(3): 711-722 and Démoulins et al., J Control Release. (2017) November 28; 266: 256-271, which can be a nanoparticle. In some embodiments, the LNP comprises or is synthesized from an ionizable lipid, a phospholipid, cholesterol, a PEGylated lipid, or any combination thereof. In some embodiments, the LNP comprises or is synthesized from the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0501] In some embodiments, the PNPs comprise or are synthesized from a non-degradable polymer, a degradable polymer, a natural material, a synthetic material, a monomer, a dendrimer, or any combination thereof.

[0502] In some embodiments, the PNP comprises or is synthesized from poly(ethylene glycol) (PEG), poly(dimethylsiloxane) (PDMS), polyethyleneimine (PEI), poly(amidoamine) (PAMAM), poly(dimethylaminoethyl)acrylate (pDMAEA), orthenine derived dendrimers, or any combination thereof. In some embodiments, the PNP comprises or is synthesized from a mannosylated-PEI polyplex. In some embodiments, the PNP comprises or is synthesized from a bioreducible linear cationic polymer (pABOL).

[0503] In some embodiments, the nanoemulsion is a water-in-oil emulsion.In some embodiments, the nanoemulsion comprises squalene, sorbitan trioleate, polysorbate 80, DOTAP, or any combination thereof.

[0504] In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435, or encoding one or more fusion proteins comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435.

[0505] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises two or more polynucleotides encoding two or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the following amino acid sequence: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 350 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 369 and 370, SEQ ID NOs: 371 and 372, SEQ ID NOs: 373 and 374, SEQ ID NOs: 375 and 376, SEQ ID NOs: 377 and 378, SEQ ID NOs: 379 and 380, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 431, SEQ ID NOs: 431 and 432, NO: 430 and 359, SEQ ID NO: 430 and 360, SEQ ID NO: 430 and 361, SEQ ID NO: 430 and 362, SEQ ID NO: 407 and 409, SEQ ID NO: 407 and 408, SEQ ID NO: 408 and 410; or SEQ ID NO: 409 and 410.

[0506] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO:527.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 4 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 3 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 2 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 527.

[0507] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 85% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 90% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 97% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 98% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 99% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein that is at least 100% identical to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 15 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 10 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 5 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO:528.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 4 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 3 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 2 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide encoding a fusion protein having 1 or fewer amino acid mismatches with the amino acid sequence of SEQ ID NO: 528.

[0508] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises one or more polynucleotides encoding one or more fusion proteins that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, and 430-435.

[0509] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0510] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0511] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0512] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0513] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0514] In various embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that has 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520.In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0515] In various embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 85% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 97% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 98% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 99% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that has 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that has 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 10 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 5 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 4 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 3 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 2 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521.In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide having 1 or fewer nucleotide mismatches with the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 521.

[0516] Composition

[0517] Provided are compositions, such as pharmaceutical compositions or immunogenic compositions, comprising one or more fusion polypeptides as described herein, or polynucleotides encoding one or more fusion polypeptides as described herein, or expression vectors, viral expression vectors or viral vectors comprising one or more such polynucleotides, or LNPs, PNPs or nanoemulsions comprising one or more such polynucleotides. The composition may also comprise a pharmaceutically acceptable diluent, carrier or excipient. Generally speaking, the compositions described herein are immunogenic compositions. In certain embodiments, the composition comprises a therapeutically effective amount of one or more fusion polypeptides as described herein, or polynucleotides encoding one or more fusion polypeptides as described herein, or expression vectors, viral expression vectors or viral vectors comprising one or more such polynucleotides, or LNPs, PNPs or nanoemulsions comprising one or more such polynucleotides.

[0518] Various pharmaceutically acceptable diluents, carriers, and excipients, as well as techniques for preparing and using pharmaceutical compositions, will be known to those skilled in the art in light of the present disclosure. Exemplary pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described, for example, in Loyd V. Allen Jr (ed.), "Remington: The Science and Practice of Pharmacy," 22nd ed., 2012, Pharmaceutical Press; Brunton, Knollman and Hilal-Dandan, "Goodman and Gilman's The Pharmacological Basis of Therapeutics," 13th ed., 2017, McGraw-Hill Education / Medical; McNally and Hastedt (eds.), "Protein Formulation and Delivery," 2nd ed., 2007, CRC Press; Banga, "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems," 3rd ed., 2015, CRC Press; Lars Hovgaard, Frokjaer and van de Weert (eds.), "Pharmaceutical Formulation Development of Peptides and Proteins," 2nd ed., 2012, CRC Press Press; Carpenter and Manning (Editors), "Rational Design of Stable Protein Formulations: Theory and Practice," 2002, Springer (Pharmaceutical Biotechnology (Book 13)); Meyer (Editor), "Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic," 2012, Woodhead Publishing.

[0519] In embodiments where the fusion polypeptide is expressed from a viral expression vector, the viral expression vector can be formulated for the desired route of administration, e.g., as an isotonic pharmaceutically acceptable aqueous solution for intravenous, intramuscular, subcutaneous, or intradermal administration. In some embodiments, the viral expression vector can be formulated for mucosal (e.g., buccal, intranasal, or intrarectal) delivery. Exemplary formulations of viral expression vectors that can be used in the pharmaceutical compositions and methods described herein are described, for example, in Manfredsson and Benskey, eds., “Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Biology),” 2019, Book 1937 in Methods in Molecular Biology Series, Humana Press; WO 2017 / 013169 (formulation of Adenoviral vectors in an aqueous mixture or freeze-dried composition in the presence of amorphous sugar and low salt concentration); and Kumru et al., J Pharm Sci. (2018) Nov; 107(11): 2764-2774 (aqueous formulations buffered in Tris and containing proline, lactose, and mannitol as stabilizing additives). The formulation of arenavirus vectors is described, for example, in WO 2009 / 083210, WO 2016 / 075250, and WO 2017 / 198726. In certain embodiments, viral expression vectors are delivered via microneedle-mediated delivery, for example, as described in Zaric et al., Expert Opin Drug Deliv. (2017) Oct; 14(10): 1177-1187. Intranasal viral vaccination by administering viral particles to the nostrils is performed, for example, as described in Dorta-Estremera et al., PLoS One. 2017 Dec 8; 12(12): e0188807. Intrarectal viral vaccination by administering viral particles to the rectum is performed, for example, as described in Patterson et al., Clin Vaccine Immunol. (2012) May; 19(5): 629-37.

[0520] In some embodiments, each carrier, diluent or excipient is "acceptable" in the sense that it is compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Typically, a pharmaceutically acceptable carrier is an aqueous pH buffer. Some examples of materials that can be used as pharmaceutically acceptable carriers, diluents or excipients include: water; buffers, e.g., buffers having a pKa in the range of about 6.0 to about 8.0, e.g., physiologically acceptable buffers, e.g., selected from phosphate, carbonate, bicarbonate, citrate, maleate, glycine-glycine, HEPES, HEPPSO, HEPPS, imidazole, BICINE, TRICINE, Tris and BIS-Tris; sugars, such as lactose, trehalose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered Tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Hank's solution, Ringer's solution; ethanol; phosphate buffered saline; amino acids (e.g., charged amino acids, including but not limited to aspartic acid, asparagine, glutamic acid, glutamine, histidine, arginine, lysine); and other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants may also be present in the composition. Solid and semisolid formulations that can be used for intravaginal or intrarectal (e.g., in the form of lozenges, pessaries, or suppositories) delivery of viral expression vectors, virions, or virus-like particles (VLPs) are described, for example, in Brown et al., PLoS One. 2017 Aug 17;12(8):e0183510; Brown et al., PLoS One. 2016 Mar 10;11(3):e0151184; and Amacker et al., npj Vaccines 5, 41 (2020).

[0521] In one particular formulation, the adenoviral vectors described herein (e.g., ChAd vectors) are formulated in an isotonic aqueous solution comprising a biocompatible buffer (e.g., HEPES and NaCl) having a neutral or near-neutral pH and a pKa in the range of about 6.0 to about 8.0, and a nonionic surfactant (e.g., F68 (also known as Poloxamer 188). In one particular formulation, the arenavirus vectors described herein (e.g., LCMV or Pichindrin mammalian arenavirus vectors) are formulated in an isotonic aqueous solution comprising HEPES buffer at pH 7.4, NaCl, and F68 (also known as Poloxamer 188). Schleiss et al. (Clin Vaccine Immunol. 2017 Jan 5;24(1):e00300-16) describe the formulation of LCMV vectors in a diluent of 25 mM HEPES, 150 mM NaCl, 0.01% PLURONIC F68 at pH 7.4, which can be used to formulate the arenavirus vectors described herein. Sorbitol was added to a final concentration of 10% before freezing below -60°C.

[0522] In another specific formulation, the adenoviral vector described herein (e.g., ChAd vector) is formulated in an aqueous buffer containing 5 mM Tris, 5% sucrose, 75 mM sodium chloride, and 1 mM magnesium chloride at pH 8.0. In some embodiments, the nominal concentration of the adenoviral vector is 5×10 11 vp / mL. In some embodiments, the adenoviral vector comprises the nucleotide sequence of SEQ ID NO: 520. In some embodiments, the adenoviral vector comprises the nucleotide sequence of SEQ ID NO: 521. In some embodiments, the adenoviral vector comprises an immunogenic polypeptide encoded by the nucleotide sequence of any one of SEQ ID NOs: 524 to 526. In some embodiments, the adenoviral vector comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526. In some embodiments, the adenoviral vector comprises an immunogenic nucleotide sequence that encodes the immunogenic polypeptide of any one of SEQ ID NOs: 527 to 528. In some embodiments, the adenoviral vector comprises a polynucleotide encoding a polypeptide sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the polypeptide sequences of SEQ ID NOs: 527 to 528, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequence of SEQ ID NOs: 527 to 528.

[0523] In another specific formulation, the SAM described herein is formulated in a LNP suspension in an aqueous buffer at pH 8.0 containing 5 mM Tris, 10% sucrose, and 10% maltose. The product is formulated to deliver 0.2 mg of SAM per mL of suspension. In some embodiments, the SAM comprises the nucleotide sequence of SEQ ID NO: 522. In some embodiments, the SAM comprises the nucleotide sequence of SEQ ID NO: 523. In some embodiments, the SAM comprises an immunogenic polypeptide encoded by the nucleotide sequence of any one of SEQ ID NOs: 524 to 526. In some embodiments, the SAM comprises a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526, wherein the percent identity is across the entire length of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526. In some embodiments, the SAM comprises an immunogenic nucleotide sequence that encodes an immunogenic polypeptide of any one of SEQ ID NOs: 527 to 528. In some embodiments, the SAM comprises a polynucleotide encoding a polypeptide sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the polypeptide sequences of SEQ ID NOs: 527 to 528, wherein the percent identity spans the entire length of the HIV immunogen nucleic acid sequence of SEQ ID NOs: 527 to 528.

[0524] The preparation and delivery method of the pharmaceutical composition will generally be adjusted according to the position to be treated and the disease. Exemplary preparations include, but are not limited to, those suitable for parenteral administration (e.g., intravenous, intraarterial, intramuscular or subcutaneous administration), including preparations encapsulated in micelles, liposomes or drug release capsules (incorporated into the active agent in the biocompatible coating designed for slow release); ingestible preparations; preparations for topical use, such as creams, ointments and gels; and other preparations, such as inhalants, aerosols and sprays. In some embodiments, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous or oral) administration. In some embodiments, the pharmaceutical composition is formulated for mucosal (e.g., buccal, intranasal, rectal and / or vaginal) administration. [052...

Claims

1. A self-amplifying RNA (SAM or saRNA), comprising: a. A polynucleotide containing the nucleic acid sequence of any one of SEQ ID NOs: 522-523; b. a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 522-523, wherein the percent identity spans the entire length of the nucleic acid sequence of SEQ ID NOs: 522-523; c. A polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 524-526; d. a polynucleotide comprising a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity spans the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526; e. a polynucleotide encoding the amino acid sequence of any one of SEQ ID NOs: 527-528; f. a polynucleotide encoding a polypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity spans the entire length of the amino acid sequence of SEQ ID NOs: 527-528; g. (i) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (ii) a promoter sequence; (iii) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (iv) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528; (v) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (vi) a polyA region; or h. (i) a first polynucleotide sequence comprising nucleotides 1 to 7512 of SEQ ID NO: 522; (ii) a promoter sequence; (iii) a second polynucleotide sequence comprising nucleotides 7537 to 7570 of SEQ ID NO: 522; (iv) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (v) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (vi) a polyA region.

2. The SAM of claim 1(h)(iv), wherein the third polynucleotide sequence encodes two or more polypeptide fragments.

3. The SAM of claim 1(h)(iv), wherein the third polynucleotide sequence encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NOs: 1-344.

4. A SAM according to claim 2 or 3, wherein: a. one or more of the polypeptide fragments are adjacent to or fused with adjacent polypeptide fragments; b. One or more of the polypeptide fragments are bound to an adjacent polypeptide fragment via one or more peptide linkers.

5. The SAM of claim 4, wherein the one or more peptide linkers are selected from one or more of the following: a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linking sequence, and combinations thereof.

6. The SAM of claim 5, wherein: a. The polyalanine linker comprises 2 or 3 consecutive alanine residues, or consists of 2 or 3 consecutive alanine residues, such as AA, AAA, AAY or AAX, wherein X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y); b. The flexible linker or polyglycine linker comprises GG, GGS, GSG or GGGS, or consists of GG, GGS, GSG or GGGS (SEQ ID NO: 421); c. The cleavable linker is selected from a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and trichothivirus (T2A)), a furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), a Nef linker sequence, and combinations, derivatives or variants thereof; d. the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382) and RRKR (SEQ ID NO: 383); e. the cleavable linker comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388); f. the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387) or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388); g. the Nef linker sequence comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390) or GALDI (SEQ ID NO: 391), wherein the percent identity spans the full length of SEQ ID NOs: 389-391; and / or h. The Nef linker sequence comprises an amino acid sequence selected from the group consisting of VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390) and GALDI (SEQ ID NO: 391).

7. The SAM according to any one of claims 1 to 6, wherein the promoter sequence comprises the polynucleotide sequence SEQ ID NO:

529.

8. An expression cassette comprising the SAM of any one of claims 1 to 7, said SAM being operably linked to one or more regulatory sequences.

9. The expression cassette of claim 8, wherein the polynucleotide is operably linked to and under the control of a constitutive promoter.

10. The expression cassette of claim 8, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, an EF1a promoter, and a 26S subgenomic promoter.

11. The expression cassette according to any one of claims 8 to 10, wherein the promoter comprises any one of the promoter sequences SEQ ID NOs: 529-530.

12. A lipid nanoparticle (LNP) comprising the SAM of any one of claims 1 to 7, or the expression cassette of any one of claims 8 to 11.

13. The LNP of claim 12, wherein the LNP comprises or is synthesized from an ionizable lipid, a phospholipid, cholesterol, a pegylated lipid, or any combination thereof.

14. The LNP according to claim 12 or 13, wherein the LNP comprises the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or is synthesized therefrom.

15. A polymer nanoparticle (PNP) comprising the SAM of any one of claims 1 to 7, or the expression cassette of any one of claims 8 to 11.

16. The PNP of claim 15, wherein the PNP comprises or is synthesized from a non-degradable polymer, a degradable polymer, a natural material, a synthetic material, a monomer, a dendrimer, or any combination thereof.

17. The PNPs of claim 15 or 16, wherein the PNPs comprise or are synthesized from poly(ethylene glycol) (PEG), poly(dimethylsiloxane) (PDMS), polyethyleneimine (PEI), poly(amidoamine) (PAMAM), poly(dimethylaminoethyl)acrylate (pDMAEA), orthenine-derived dendrimers, or any combination thereof.

18. The PNP according to any one of claims 15 to 17, wherein the PNP comprises a mannosylated-PEI polyplex, or is synthesized therefrom.

19. The PNP according to any one of claims 15 to 18, wherein the PNP comprises, or is synthesized from, a bioreducible linear cationic polymer (pABOL).

20. A nanoemulsion comprising the SAM of any one of claims 1 to 7, or the expression cassette of any one of claims 8 to 11.

21. The nanoemulsion of claim 20, wherein the nanoemulsion is a water-in-oil emulsion.

22. The nanoemulsion of claim 20, wherein the nanoemulsion comprises squalene, sorbitan trioleate, polysorbate 80, DOTAP, or any combination thereof.

23. An expression vector comprising the SAM of any one of claims 1 to 7, or the expression cassette of any one of claims 8 to 10.

24. The expression vector according to claim 23, wherein the expression vector is a plasmid vector, a bacterial vector or a viral expression vector.

25. The expression vector according to claim 23 or 24, wherein the expression vector is a viral expression vector.

26. The expression vector of claim 25, wherein the viral expression vector is a DNA virus or an RNA virus.

27. The expression vector of any one of claims 25 to 26, wherein the viral expression vector is replication-defective, replication-deficient, replication-attenuated or replication-competent.

28. The expression vector of any one of claims 25 to 27, wherein the viral expression vector is from a virus selected from the group consisting of adenovirus, adeno-associated virus, arenavirus, alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus, and vaccinia virus.

29. The expression vector of any one of claims 25 to 28, wherein the viral expression vector is from a virus from a taxonomic family selected from the group consisting of Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., Modified Vaccinia Virus Ankara (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus, e.g., Venezuelan equine encephalitis virus).

30. The expression vector of any one of claims 25 to 29, wherein the viral expression vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

31. The expression vector of claim 30, wherein the viral expression vector is an arenavirus vector selected from the group consisting of lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus).

32. The expression vector of claim 30 or 31, wherein the arenavirus vector comprises a bi-segmented genome.

33. The expression vector of claim 30 or 31, wherein the arenavirus vector comprises a three-segmented genome.

34. The expression vector of any one of claims 25 to 29, wherein the viral expression vector is a human adenovirus or a simian adenovirus.

35. The expression vector of claim 34, wherein the simian adenovirus is selected from the group consisting of a chimpanzee adenovirus, a gorilla adenovirus, and a rhesus monkey adenovirus.

36. The expression vector of any one of claims 25 to 29 and 34, wherein the viral expression vector is an adenovirus vector selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (ChAd), gorilla adenovirus, and rhesus monkey adenovirus.

37. The expression vector of claim 36, wherein the ChAd is selected from the group consisting of ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22), ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (Ad C28), ChAd30 (AdC30), ChAd31 (AdC31), ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 (AdC55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAd143 (AdC143), ChAd144 (AdC144), ChAd145 (AdC145) and ChAd147 (AdC147).

38. The expression vector of claim 37, wherein the ChAd is ChAd68.

39. The expression vector of claim 36, wherein the gorilla adenovirus is selected from the group consisting of GC44, GC45 and GC46.

40. The expression vector of claim 36, wherein the rhesus adenovirus (RhAd) is selected from the group consisting of RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, and RhAd66.

41. The expression vector of any one of claims 34 to 40, wherein the human adenovirus or simian adenovirus comprises a modification of one or more adenoviral genes.

42. The expression vector of claim 41, wherein the one or more adenoviral genes are selected from E1, E3 and E4 ORF2-4.

43. The expression vector according to claim 41 or 42, wherein the modification comprises deleting or substituting one or more nucleotides in the one or more adenoviral genes, or inserting one or more nucleotides into the one or more adenoviral genes.

44. The expression vector of any one of claims 41 to 43, wherein the human adenovirus or simian adenovirus comprises a deletion of at least a portion of the E1 region.

45. The expression vector of claim 44, wherein the polynucleotide is inserted into the deletion of at least a portion of the E1 region.

46. ​​The expression vector according to any one of claims 23 to 45, wherein the expression vector comprises any one of the nucleic acid sequences SEQ ID NOs: 520-521.

47. An expression vector according to any one of claims 23 to 45, wherein the expression vector comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences SEQ ID NOs: 520-521, wherein the percent identity spans the entire length of the nucleic acid sequence SEQ ID NOs: 520-521.

48. An expression vector comprising: a. nucleic acid sequence of any one of SEQ ID NOs: 524-526; b. a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity spans the entire length of the nucleic acid sequence of SEQ ID NOs: 524-526; c. a polynucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 527-528; d. a polynucleotide sequence encoding a polynucleotide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity spans the entire length of the amino acid sequence of SEQ ID NOs: 527-528; e. (i) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (ii) a promoter sequence; (iii) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (iv) a third polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528, wherein the percent identity is across SEQ ID NOs: NO: 345-371, 373-377, 407-411, 422-423, 430-435, 527 and 528; (v) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (vi) a polyA sequence; and (vii) a fifth polynucleotide sequence comprising nucleotides 4620 to 32350 of SEQ ID NO: 520; or f. (i) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (ii) a promoter sequence; (iii) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (iv) a third polynucleotide sequence encoding one or more polypeptide fragments, wherein the one or more polypeptide fragments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-344, wherein the percent identity spans the entire length of SEQ ID NOs: 1-344; (v) a fourth polynucleotide sequence comprising nucleotides 4352 to 4395 of SEQ ID NO: 520; (vi) a polyA sequence; and (vii) a fifth polynucleotide sequence comprising nucleotides 4620 to 32350 of SEQ ID NO:

520.

49. The expression vector of claim 48, wherein the third polynucleotide sequence encodes two or more polypeptide fragments.

50. The expression vector of claim 48, wherein the third polynucleotide sequence encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more polypeptide fragments selected from SEQ ID NOs: 1-344.

51. The expression vector of claim 49 or 50, wherein one or more of the polypeptide fragments are contiguous or fused to an adjacent polypeptide fragment.

52. The expression vector of claim 49 or 50, wherein one or more of the polypeptide fragments are bound to adjacent polypeptide fragments via one or more peptide linkers.

53. The expression vector of claim 52, wherein the one or more peptide linkers are selected from one or more of the following: a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linking sequence, and combinations thereof.

54. The expression vector according to claim 53, wherein: a. The polyalanine linker comprises 2 or 3 consecutive alanine residues, or consists of 2 or 3 consecutive alanine residues, such as AA, AAA, AAY or AAX, wherein X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y); b. The flexible linker or polyglycine linker comprises GG, GGS, GSG or GGGS, or consists of GG, GGS, GSG or GGGS (SEQ ID NO: 421); c. The cleavable linker is selected from a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and trichothivirus (T2A)), a furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), a Nef linker sequence, and combinations, derivatives or variants thereof; d. the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382) and RRKR (SEQ ID NO: 383); e. the cleavable linker comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388), wherein the percent identity spans the entire length of SEQ ID NOs: 384-388; f. the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 384), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 385), RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 386), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 387) or EGRGSLLTCGDVEENPGP (SEQ ID NO: 388); g. the Nef linker sequence comprises or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390) or GALDI (SEQ ID NO: 391), wherein the percent identity spans the full length of SEQ ID NOs: 389-391; and / or h. The Nef linker sequence comprises an amino acid sequence selected from the group consisting of VHAGPIA (SEQ ID NO: 389), VHAGPVA (SEQ ID NO: 390) and GALDI (SEQ ID NO: 391).

55. The expression vector according to any one of claims 48 to 54, wherein the promoter sequence comprises the polynucleotide sequence of SEQ ID NO:

529.

56. A viral vector comprising the SAM of any one of claims 1 to 7, the expression cassette of any one of claims 8 to 10, or the expression vector of any one of claims 23 to 55.

57. The viral vector of claim 56, wherein: a. The viral vector is derived from or based on a DNA virus or an RNA virus; b. The viral vector is replication-deficient, replication-deficient, replication-attenuated or replication-competent; c. The viral vector is from a virus selected from the group consisting of adenovirus, adeno-associated virus, arenavirus, alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus and vaccinia virus; d. The viral vector is derived from or based on a virus from a taxonomic family selected from the group consisting of: Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., Modified Vaccinia Ankara virus (MVA)), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Maraba vesiculovirus), Togaviridae (e.g., alphavirus, e.g., Venezuelan equine encephalitis virus); e. The viral vector is derived from or based on an arenavirus vector selected from the group consisting of: lymphocytic choriomeningitis mammalian arenavirus (LCMV), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV) and Whitewater Arroyo virus (WWAV); or f. The viral vector is derived from or based on an arenavirus vector selected from the group consisting of: lymphocytic choriomeningitis mammalian arenavirus (LCMV) or Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus or Pichinde arenavirus).

58. The viral vector of claim 57, wherein the arenavirus vector comprises a two-segmented genome or a three-segmented genome.

59. The viral vector of any one of claims 56 or 57, wherein the viral vector is a human adenovirus or a simian adenovirus.

60. The viral vector of claim 59, wherein the simian adenovirus is selected from the group consisting of a chimpanzee adenovirus, a gorilla adenovirus, and a rhesus monkey adenovirus.

61. The viral vector of any one of claims 56, 57 and 59, wherein the viral vector is derived from or based on an adenovirus vector selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (ChAd), gorilla adenovirus and rhesus monkey adenovirus.

62. The viral vector of claim 61, wherein: a. The ChAd is selected from the group consisting of ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22), ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (AdC28), ChAd3 0 (AdC30), ChAd31 (AdC31), ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 (AdC55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAd143 (AdC143), ChAd144 (AdC144), ChAd145 (AdC145), and ChAd147 (AdC147); b. The gorilla adenovirus is selected from GC44, GC45 and GC46; c. The rhesus monkey adenovirus (RhAd) is selected from RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65 and RhAd66.

63. The viral vector of claim 62, wherein the ChAd is ChAd68.

64. The viral vector of any one of claims 59 to 63, wherein the human adenovirus or simian adenovirus comprises a modification of one or more adenoviral genes.

65. The viral vector of claim 64, wherein the one or more adenoviral genes are selected from E1, E3 and E4 ORF2-4.

66. The viral vector according to claim 64 or 65, wherein: a. the modification comprises deleting or replacing one or more nucleotides in the one or more adenoviral genes, or inserting one or more nucleotides into the one or more adenoviral genes; or b. The human adenovirus or simian adenovirus comprises a deletion of at least a part of the E1 region.

67. The viral vector of claim 66.a, wherein the polynucleotide is inserted into the deletion of at least a portion of the E1 region.

68. The viral vector of any one of claims 56 to 67, wherein the viral vector comprises any one of the nucleic acid sequences SEQ ID NOs: 520-521.

69. A viral vector according to any one of claims 56 to 67, wherein the vector comprises a nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the nucleic acid sequences SEQ ID NOs: 520-521, wherein the percent identity spans the entire length of the nucleic acid sequence SEQ ID NOs: 520-521.

70. A host cell comprising one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

71. The host cell of claim 70, wherein the one or more polynucleotides are not integrated into the host cell genome, e.g., are episomal.

72. The host cell of claim 70, wherein the one or more polynucleotides are integrated into the host cell genome.

73. A host cell according to any one of claims 70 to 72, wherein the host cell is a mammalian cell.

74. The host cell of claim 73, wherein the mammalian cell is a human cell.

75. The host cell of claim 73, wherein the mammalian cell is not a human cell.

76. A host cell according to any one of claims 70 to 75, wherein the host cell is in vitro.

77. A host cell according to any one of claims 70 to 75, wherein the host cell is in vivo.

78. A polypeptide comprising: a. HIV immunogen amino acid sequence SEQ ID NO: 527-528; or b. an amino nucleic acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528, wherein the percent identity is across the entire length of the HIV immunogen amino acid sequences SEQ ID NOs: 527-528.

79. A composition comprising: (a) one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, one or more viral vectors of any one of claims 56 to 69, or one or more polypeptides of claim 78; and (b) a pharmaceutically acceptable diluent, carrier, or excipient.

80. The composition of claim 79, comprising two or more SAMs of any one of claims 1 to 7, two or more expression cassettes of any one of claims 8 to 11, two or more LNPs of any one of claims 12 to 14, two or more PNPs of any one of claims 15 to 19, two or more nanoemulsions of any one of claims 20 to 22, two or more expression vectors of any one of claims 23 to 55, two or more viral vectors of any one of claims 56 to 69, or two or more polypeptides of claim 78.

81. A composition comprising: (a) one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, or one or more nanoemulsions of any one of claims 20 to 22; and (b) one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

82. A composition comprising: (a) one or more polypeptides of claim 78; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

83. The composition of claim 82, comprising two or more polypeptides of claim 78.

84. The composition of claim 81, further comprising a pharmaceutically acceptable diluent, carrier or excipient.

85. The composition of any one of claims 79 to 84, further comprising one or more of an adjuvant, an immunostimulant, a detergent, a micelle former, and an oil.

86. The composition of claim 85, wherein the immunostimulatory agent is selected from a toll-like receptor (TLR) agonist, a cytokine, a non-coding immunostimulatory polynucleotide, an inhibitor of an inhibitory immune checkpoint protein, or a stimulator of a stimulatory immune checkpoint protein.

87. The composition of claim 86, wherein: a. The cytokine is selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-ɑ, IFN-γ, GM-CSF, FLT3LG, and combinations and functional variants thereof; or b. The non-coding immunostimulatory polynucleotide is selected from pathogen-activated molecular patterns (PAMPs), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides and immunostimulatory RNA (isRNA, such as CV8102).

88. according to the compositions described in any one of claims 79 to 87, wherein said compositions is formulated for use via the approach selected from the group consisting of: intravenous, intramuscular, intradermal, subcutaneous and mucosal (e.g., buccal, intranasal, intrarectal, intravaginal).

89. A composition according to any one of claims 79 to 87.a, wherein the composition is formulated as a liquid.

90. The composition of any one of claims 79 to 87.a, wherein the composition is lyophilized.

91. A kit comprising one or more components selected from the group consisting of one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, one or more viral vectors of any one of claims 56 to 69, or one or more polypeptides of claim 78.

92. A kit comprising: (a) a first component selected from one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, or one or more nanoemulsions of any one of claims 20 to 22; and (b) a second component comprising one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

93. A kit comprising one or more unit doses of the following components: one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, one or more viral vectors of any one of claims 56 to 69, or one or more polypeptides of claim 78.

94. A kit comprising: (a) one or more unit doses of one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, or one or more nanoemulsions of any one of claims 20 to 22; and (b) one or more unit doses of one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

95. A kit comprising one or more unit doses of one or more polypeptides of claim 78.

96. The kit of claim 91, wherein: a. said one or more components are in a single container; b. the one or more components are in two or more separate containers; c. The first component and the second component are in a single container; or d. The first component and the second component are in two or more separate containers.

97. The kit of claim 93 or 95, wherein: a. said one or more unit doses are in a single container; b. The one or more unit doses are in two or more separate containers.

98. The kit of any one of claims 96 to 97, comprising one or more containers selected from the group consisting of a vial, an ampoule, and a prefilled syringe.

99. The kit of any one of claims 96 to 98, comprising one or more containers comprising the one or more SAMs, one or more LNPs, one or more PNPs, one or more nanoemulsions, one or more carriers, or one or more polypeptides in an aqueous solution.

100. The kit according to any one of claims 93, 94, 95 and 97 to 99, wherein: (a) the one or more unit doses are the same; or (b) the one or more unit doses are different.

101. A kit according to any one of claims 93, 94, 95 and 97 to 100, comprising one or more unit doses of one or more viral vectors of any one of claims 23 to 47, wherein the unit dose is about 10 3 About 10 15 The concentration of the virus is in the range of 1 focus forming unit (FFU) or 1 plaque forming unit (PFU) or 1 infectious unit (IU) or 1 virus particle (vp).

102. The kit of claim 101, wherein the unit dose of the one or more viral vectors is about 10 4 About 10 7 Virus FFU or PFU or IU or vp.

103. The kit of claim 101, wherein the unit dose of the one or more viral vectors is about 10 3 About 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 or 10 15 Virus FFU or PFU or IU or vp.

104. The kit of any one of claims 93, 94, and 97 to 100, comprising one or more unit doses of one or more SAMs of any one of claims 1 to 7, wherein the unit dose is in the range of about 1 μg to about 1000 μg.

105. The kit of claim 104, wherein the unit dose of the one or more SAMs is from about 15 μg to about 500 μg.

106. The kit of claim 104, wherein the unit dose of the one or more SAMs is from about 1 μg to about 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg.

107. The composition of any one of claims 79 to 90 or the kit of any one of claims 91 to 106, further comprising one or more unit doses of one or more additional therapeutic agents.

108. A composition or kit according to claim 107, wherein the one or more additional therapeutic agents are selected from one or more agents that activate latent HIV, one or more agonists or activators of one or more toll-like receptors (TLRs), one or more interleukin receptor agonists, one or more cytokines, one or more receptor agonists, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors, one or more agonists, activators or stimulators of T cell stimulatory immune checkpoint proteins or receptors, one or more CD47 inhibitors, one or more immune-based therapies, one or more broadly neutralizing antibodies (bnAbs), and one or more antiviral agents.

109. The composition or kit of claim 108, wherein: a. The one or more agents that activate latent HIV are one or more latency reversal agents (LRA); b. The TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist and a TLR9 agonist.

110. The composition or kit of claim 109, wherein the one or more LRAs are selected from the group consisting of: an agonist or activator of one or more toll-like receptors (TLRs), a histone deacetylase (HDAC) inhibitor, a proteasome inhibitor, a protein kinase C (PKC) activator, a Smyd2 inhibitor, a BET-bromodomain 4 (BRD4) inhibitor, ionomycin, an inhibitor of apoptosis protein (IAP) antagonist, and a second mitochondrial-derived caspase activator (SMAC) mimetic.

111. The composition or kit of claim 109, wherein: a. the TLR7 agonist is selected from the group consisting of GS 9620 (visamod), R848 (resiquimod), DS-0509, LHC-165 and TMX-101 (imiquimod), and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688, R848 (resiquimod), CV8102 (dual TLR7 / TLR8 agonist) and NKTR-262 (dual TLR7 / TLR8 agonist); or b. The TLR9 agonist is selected from the group consisting of: AST-008, cobitomot, CMP-001, IMO-2055, IMO-2125, linimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatomod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefetomod (MGN-1703), CYT-003, CYT-003-QbG10, tesotommod and PUL-042.

112. The composition or kit of claim 111, further comprising GS 9620 (visamod).

113. The composition or kit of claim 111, further comprising lefetomod (MGN-1703).

114. The composition or kit of claim 108, wherein: a. The interleukin receptor agonist is an interleukin receptor agonist selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG; b. The one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG, and combinations and functional variants thereof; c. The receptor agonist is an agonist of one or more receptors selected from the group consisting of: fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain-containing protein 2 (NOD2); The one or more inhibitors of dT cell inhibitory immune checkpoint proteins or receptors inhibit checkpoint proteins or receptors selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (B =TLA); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); The one or more agonists, activators or stimulators of eT cell stimulatory immune checkpoint proteins or receptors agonize, activate or stimulate T cell stimulatory immune checkpoint proteins or receptors selected from the group consisting of: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); f. the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors and capsid inhibitors; or g. The one or more immune-based therapies are selected from the group consisting of interferon alpha, interferon alpha-2b, interferon alpha-n3, pegylated interferon alpha, interferon gamma, fms-related tyrosine kinase 3 (FLT3) agonists, gepon, nomofuron, pegylated interferon alpha-2a, pegylated interferon alpha-2b and RPI-MN.

115. The composition or kit of claim 114, wherein the CTLA4 inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155 , KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155(CTLA4 / PD-L1 / CD28), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA4 / PD-1) and BPI-002.

116. The composition or kit of claim 108, 114 or 115, further comprising ipilimumab.

117. The composition or kit of claim 114, wherein the PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplizumab, pidilizumab, AB122 (sepalizumab), AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalizumab), JNJ-63723283, genol monoclonal antibody (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalimumab), MSB-2311, JTX-4014, BGB-A333, S HR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1) MGD-019 (PD-1 / CTLA4), KN-046 ( , MEDI-5752 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFb-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), INBRX-105 (4-1BB / PDL1), GS-4224, GS-4416, INCB086550, and MAX10181.

118. The composition or kit of claim 108, 114 or 117, further comprising pembrolizumab.

119. The composition or kit of claim 108, 114 or 117, further comprising nivolumab.

120. The composition or kit of claim 108, 114 or 117, further comprising AB122 (sepalizumab).

121. The composition or kit of claim 114, wherein the FLT3 agonist is selected from GS-3583 and CDX-301.

122. The composition or kit of claim 108, 114 or 121, further comprising GS-3583.

123. The composition or kit of claim 108, 114 or 121 further comprising CDX-301.

124. The composition or kit of claim 108, further comprising one or more bnAbs.

125. The composition or kit of claim 108, wherein the one or more bnAbs are HIV bnAbs.

126. A composition or kit according to claim 125, wherein the HIV bnAb is highly effective against most prevalent HIV strains, neutralizes a wide range of genetically diverse HIV-1 subtypes, and / or potently neutralizes a significant proportion of primary isolates, or exhibits some ability to span evolutionary branches, but has difficulty neutralizing Tier 2 and Tier 3 viruses.

127. The composition or kit of any one of claims 108 and 124 to 126, wherein the bnAb is directed to the surface of Env: the CD4 binding site (CD4bs), N-glycans associated with the V1 / V2 and V3 loops, the silent face of gp120, the membrane proximal external region (MPER) of gp41, or a larger site spanning the interface between gp41 and gp120.

128. according to the composition or kit described in any one of claims 108 and 124 to 127, wherein the bnAb is selected from 10-1074, 10E8, 12A12, 12A21, 2F5, 2G12, 3BC176, 3BNC117, 3BNC55, 3BNC60, 3BNC62, 447-52D, 4E10, 5H / I1-BMV-D5, 8ANC195, b12, CHO1, CH02, CH03, CH04, CH103, HGN194, HJ16, HK20, M66.6, NIH45-46, P G16, PG9, PGT121, PGT122, PGT123, PGT125, PGT126, PGT127, PGT128, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, PGT144, PGT145, VRC-CH30, CRC-CH31, CRC-CH32, CRC-CH33, CRC-CH34, VRC-PG04, VRC-PG04b, VRC01, VRC02, VRC03 and Z13.

129. The composition or kit of any one of claims 108 and 124 to 128, wherein the bnAb is an anti-CD4 bnAb.

130. The composition or kit of claim 129, wherein the anti-CD4 bnAb is selected from 3BNC117 and VRC01.

131. The composition or kit of any one of claims 108 and 124 to 128, wherein the bnAb is an anti-V3 bnAb.

132. The composition or kit of claim 131, wherein the anti-V3bnAb is 10-1074.

133. A method of eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, one or more viral vectors of any one of claims 56 to 69, one or more polypeptides of claim 78, or one or more compositions of any one of claims 79 to 90.

134. A method of treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22, one or more expression vectors of any one of claims 23 to 55, one or more viral vectors of any one of claims 56 to 69, one or more polypeptides of claim 78, or one or more compositions of any one of claims 79 to 90.

135. A method of eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject: (a) one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22; and (b) one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

136. A method of treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject: (a) one or more SAMs of any one of claims 1 to 7, one or more expression cassettes of any one of claims 8 to 11, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22; and (b) one or more expression vectors of any one of claims 23 to 55, or one or more viral vectors of any one of claims 56 to 69.

137. The method of any one of claims 133 to 136, wherein: a. The subject is infected with HIV-1, suspected of being infected with HIV-1, or at risk of being infected with HIV-1; b. The subject is chronically infected with HIV-1; c. The subject is acutely infected with HIV-1; or d. The subject has Fiebig stage IV or earlier, such as Fiebig stage III, Fiebig stage II or Fiebig stage I HIV-1 infection.

138. The method of any one of claims 133 to 137, wherein administration comprises a route of administration selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, rectal, vaginal).

139. The method of any one of claims 133 to 138, wherein the method comprises administering about 10 3 About 10 15 One or more viral vectors of any one of claims 23 to 47 are administered in a dosage range of 100 viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp).

140. The method of claim 139, wherein the one or more viral vectors are administered at about 10 4 About 10 7 Virus FFU or PFU or IU or vp, for example about 10 3 About 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 or 10 15 The dosage range is 100 virus FFU or PFU or IU or vp.

141. The method of any one of claims 133 to 140, wherein the method comprises administering one or more SAMs of any one of claims 1 to 7, one or more LNPs of any one of claims 12 to 14, one or more PNPs of any one of claims 15 to 19, one or more nanoemulsions of any one of claims 20 to 22 at a dose range of about 1 μg to about 1000 μg.

142. The method of any one of claims 133 to 141, wherein the one or more SAMs, LNPs, PNPs, or nanoemulsions are administered in a dose range of about 15 μg to about 500 μg.

143. The method of any one of claims 133 to 142, wherein the one or more SAMs, LNPs, PNPs, or nanoemulsions are administered in a dose range of about 1 μg to about 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg.

144. The method of any one of claims 133 to 143, comprising a prime-boost regimen comprising: (i) administering a priming composition at a first time point, followed by administration of one or more boosting compositions at one or more subsequent time points (e.g., prime-boost-boost-boost, etc.); (ii) one or more iterations of administering a priming composition at a first time point followed by a boosting composition at a second time point (e.g., prime-boost-prime-boost, etc.); or (iii) administering a priming composition at a first time point, administering a boosting composition at one or more subsequent time points, and administering the priming composition at a subsequent time point after administration of the boosting composition in one or more iterations (e.g., prime-boost-boost-prime, etc.).

145. The method of claim 144, wherein the administration of the priming composition and the one or more boosting compositions is separated by at least 1 week, 2 weeks, 3 weeks or 1 month, such as at least 2 months, 3 months, 4 months, 5 months or 6 months.

146. The method of any one of claims 133 to 145, wherein: a. The priming composition and the boosting composition comprise polynucleotides encoding the same immunogenic polypeptide; b. the priming composition and the boosting composition comprise polynucleotides encoding different immunogenic polypeptides; c. the priming composition and the boosting composition comprise the same one or more polypeptides, polynucleotides, SAMs, LNPs, PNPs, nanoemulsions or viral vectors; or d. The priming composition and the boosting composition comprise different polypeptides, polynucleotides, SAMs, LNPs, PNPs, nanoemulsions or viral vectors.

147. The method of claim 146, comprising priming with a first polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector, and then boosting with a second polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector.

148. The method of any one of claims 133 to 147, wherein the prime-boost regimen comprises: a. Prime with a viral vector and then boost with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA; b. priming with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA, and then boosting with a viral vector; c. priming with a first viral vector and then boosting with a second viral vector, wherein the first viral vector and the second viral vector are from the same, related or unrelated taxonomic family; d. priming with a first replication-deficient viral vector and then boosting with a second replication-deficient viral vector, wherein the first replication-deficient viral expression vector and the second replication-deficient viral expression vector are from the same, related or unrelated taxonomic family; e. priming with a first attenuated defective viral vector and then boosting with a second replicating attenuated viral vector, wherein the first replicating attenuated viral vector and the second replicating attenuated viral vector are from the same, related or unrelated taxonomic family; f. Prime with a replication-deficient viral vector and then boost with a replication-attenuated viral vector; g. Initial immunization with a replication-attenuated viral expression vector and then boosted with a replication-deficient viral vector; h. Prime with a viral vector and then boost with a composition comprising SAM; i. priming with an adenoviral vector and then boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA; j. Prime with an adenoviral vector and then boost with a composition comprising SAM; k. priming with a ChAd vector and then boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA or self-replicating RNA; or 1. Prime with ChAd vector, then boost with a composition containing SAM.

149. The method of any one of claims 133 to 148, wherein the subject is not receiving antiretroviral therapy (ART), or ART is interrupted prior to administration of the one or more polynucleotides, polypeptides, SAMs, LNPs, PNPs, nanoemulsions, expression cassettes, or viral vectors.

150. The method of any one of claims 133 to 149, wherein ART is discontinued following one or more administrations of the polynucleotide, polypeptide, SAM, LNP, PNP, nanoemulsion, expression cassette or viral vector.

151. The method of any one of claims 133 to 150, further comprising administering to the subject one or more additional therapeutic agents, eg, two, three, four or more additional therapeutic agents.

152. The method of claim 151, wherein the one or more therapeutic agents are selected from one or more agents that activate latent HIV, one or more toll-like receptor (TLR) agonists or activators, one or more interleukin receptor agonists, one or more cytokines, one or more receptor agonists, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors, one or more agonists, activators or stimulators of T cell stimulatory immune checkpoint proteins or receptors, one or more CD47 inhibitors, one or more antiviral agents, one or more immune-based therapies, and one or more broadly neutralizing antibodies (bnAbs).

153. The method of claim 152, wherein: a. The one or more agents that activate latent HIV are one or more latency reversal agents (LRA); b. the one or more TLR agonists or activators are selected from the group consisting of: a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist; c. the one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG, and combinations and functional variants thereof; d. The one or more interleukin receptor agonists are agonists of interleukins selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF and FLT3LG; e. The one or more receptor agonists are agonists of receptors selected from the group consisting of: fms-related tyrosine kinase 3 (FLT3), interferon gene stimulator (STING) receptor, DExD / H box helicase 58 (DDX58; also known as RIG-I), nucleotide binding oligomerization domain-containing protein 2 (NOD2); the one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors are inhibitors of T cell inhibitory immune checkpoint proteins or receptors selected from the group consisting of: CD274 (CD274, PD L1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing T cell activation inhibitor 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF1 1, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS 9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); The one or more agonists, activators or stimulators of fT cell stimulatory immune checkpoint proteins or receptors agonize, activate or stimulate T cell stimulatory immune checkpoint proteins or receptors selected from the following items: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); g. the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors and capsid inhibitors; or h. The one or more immune-based therapies are selected from the group consisting of: interferon alpha, interferon alpha-2b, interferon alpha-n3, pegylated interferon alpha, interferon gamma; fms-related tyrosine kinase 3 (FLT3) agonists, gepon, nomofuron, pegylated interferon alpha-2a, pegylated interferon alpha-2b and RPI-MN.

154. The method of claim 153, wherein the one or more LRAs are selected from one or more agonists or activators of toll-like receptors (TLRs), histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitor of apoptosis protein (IAP) antagonists, and a second mitochondrial-derived caspase activator (SMAC) mimetic.

155. The method of claim 153, wherein the TLR7 agonist is selected from the group consisting of GS9620 (visamod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod), and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688, R848 (resiquimod), CV8102 (dual TLR7 / TLR8 agonist), and NKTR-262 (dual TLR7 / TLR8 agonist).

156. The method of claim 155, further comprising administering GS 9620 (visamod) to the subject.

157. The method of claim 153, wherein the TLR9 agonist is selected from the group consisting of AST-008, cobitomot, CMP-001, IMO-2055, IMO-2125, linimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatomod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefetomod (MGN-1703), CYT-003, CYT-003-QbG10, tesotommod, and PUL-042.

158. The method of claim 157, further comprising administering lefetomod (MGN-1703) to the subject.

159. The method of any one of claims 152 to 158, further comprising administering one or more bnAbs to the subject.

160. The method of claim 152 or 159, wherein the one or more bnAbs are HIV bnAbs.

161. A method according to claim 160, wherein the HIV bnAb is highly effective against the most prevalent HIV strains, neutralizes a wide range of genetically diverse HIV-1 subtypes, and / or potently neutralizes a significant proportion of primary isolates, or exhibits some ability to cross evolutionary branches, but has difficulty neutralizing Tier 2 and Tier 3 viruses.

162. The method of any one of claims 152 and 159 to 161, wherein the bnAb is directed to the surface of Env: the CD4 binding site (CD4bs), N-glycans associated with the V1 / V2 and V3 loops, the silent face of gp120, the membrane proximal external region (MPER) of gp41, or a larger site spanning the interface between gp41 and gp120.

163. The method of any one of claims 152 and 159 to 162, wherein the bnAb is selected from 10-1074, 10E8, 12A12, 12A21, 2F5, 2G12, 3BC176, 3BNC117, 3BNC55, 3BNC60, 3BNC62, 447-52D, 4E10, 5H / I1-BMV-D5, 8ANC195, b12, CHO1, CH02, CH03, CH04, CH103, HGN194, HJ16, HK20, M66.6, NIH45-46, PG1 6. PG9, PGT121, PGT122, PGT123, PGT125, PGT126, PGT127, PGT128, PGT130, PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, P GT143, PGT144, PGT145, VRC-CH30, CRC-CH31, CRC-CH32, CRC-CH33, CRC-CH34, VRC-PG04, VRC-PG04b, VRC01, VRC02, VRC03 and Z13.

164. The method of any one of claims 152 and 159 to 163, wherein the bnAb is an anti-CD4 bnAb.

165. The method of claim 164, wherein the anti-CD4 bnAb is selected from 3BNC117 and VRC01.

166. The method of any one of claims 152 and 159 to 163, wherein the bnAb is an anti-V3 bnAb.

167. The method of claim 166, wherein the anti-V3 bnAb is 10-1074.

168. The method of claim 153, wherein: a. The CTLA4 inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0 161. ATOR-1144, PBI-5D3H5, FPT-155(CTLA4 / PD-L1 / CD28), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA4 ), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA4 / PD-1) and BPI-002; b. The PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of: pembrolizumab, nivolumab, cemiplizumab, pidilizumab, AB122 (sepalizumab), AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA- 012, BI-754091, AGEN-2034 (batilizumab), JS-001 (toripalizumab), JNJ-63723283, genozumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalimumab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1) MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFb-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), INBRX-105 (4-1BB / PDL1), GS-4224, GS-4416, INCB086550, and MAX10181; c. The FLT3 agonist is selected from GS-3583 and CDX-301.

169. The method of claim 152, 153, or 168, further comprising administering ipilimumab to the subject.

170. The method of claim 152, 153, or 168, further comprising administering pembrolizumab to the subject.

171. The method of claim 152, 153, or 168, further comprising administering nivolumab to the subject.

172. The method of claim 153 or 168, further comprising administering AB122 (sepalizumab) to the subject.

173. The method of claim 152, 153, or 168, wherein the method further comprises administering GS-3583 to the subject.

174. The method of claim 152, 153, or 168, wherein the method further comprises administering CDX-301 to the subject.

175. The method of any one of claims 133 to 174, wherein the subject does not show symptoms of HIV and AIDS for at least 6 months, at least 1 year, at least 2 years, at least 3 years or more without antiretroviral therapy (ART) following one or more administrations of one or more of the polynucleotides, polypeptides, SAMs, LNPs, PNPs, microemulsions, expression cassettes, or viral vectors, optionally in combination with one or more additional therapeutic agents.

176. The method of any one of claims 133 to 175, wherein the subject has a viral load of less than 500 copies per ml of blood, e.g., less than 400, less than 300, less than 200, less than 100, less than 50, for at least 6 months, at least 1 year, at least 2 years, at least 3 years or more, in the absence of antiretroviral therapy (ART), following one or more administrations of one or more of a polynucleotide, polypeptide, SAM, LNP, PNP, microemulsion, expression cassette or viral vector, optionally in combination with one or more additional therapeutic agents.

177. One or more SAMs according to any one of claims 1 to 7, one or more expression cassettes according to any one of claims 8 to 11, one or more LNPs according to any one of claims 12 to 14, one or more PNPs according to any one of claims 15 to 19, one or more nanoemulsions according to any one of claims 20 to 22, one or more expression vectors according to any one of claims 23 to 55, one or more viral vectors according to any one of claims 56 to 69, one or more polypeptides according to claim 78 or 78.a, or one or more compositions according to any one of claims 79 to 90, for use in eliciting an immune response against human immunodeficiency virus (HIV) in a subject in need thereof.

178. One or more SAMs according to any one of claims 1 to 7, one or more expression cassettes according to any one of claims 8 to 11, one or more LNPs according to any one of claims 12 to 14, one or more PNPs according to any one of claims 15 to 19, one or more nanoemulsions according to any one of claims 20 to 22, one or more expression vectors according to any one of claims 23 to 55, one or more viral vectors according to any one of claims 56 to 69, one or more polypeptides according to claim 78, or one or more compositions according to any one of claims 79 to 90, for use in treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof.

179. One or more SAMs according to any one of claims 1 to 7, one or more expression cassettes according to any one of claims 8 to 11, one or more LNPs according to any one of claims 12 to 14, one or more PNPs according to any one of claims 15 to 19, one or more nanoemulsions according to any one of claims 20 to 22, one or more expression vectors according to any one of claims 23 to 55, one or more viral vectors according to any one of claims 56 to 69, one or more polypeptides according to claim 78, or one or more compositions according to any one of claims 79 to 90, for use as a medicament.

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