HIV Immunogenic Polypeptides and Vaccines, and Their Use

Abstract

The present disclosure relates to an HIV immunogenic polypeptide, a polynucleotide encoding such an HIV immunogenic polypeptide, a self-amplifying messenger RNA (SAM) vector comprising such a polynucleotide, and a viral vector comprising such a polynucleotide. Polynucleotides comprising any one of the HIV immunogenic nucleic acid sequences of SEQ ID NOs: 524-526 are disclosed herein. Polynucleotides comprising an HIV immunogenic nucleic acid sequence encoding any one of the HIV immunogenic nucleic acid sequences of SEQ ID NOs: 527-528 are disclosed herein.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 388,599, filed Jul. 12, 2022; U.S. Provisional Patent Application No. 63 / 384,020, filed Nov. 16, 2022; and U.S. Provisional Patent Application No. 63 / 485,122, filed Feb. 15, 2023, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] (Sequence Listing) This application includes a sequence listing that has been electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy created on Jun. 27, 2023, is named 1444 - WO - PCT.xml and is 691,866 bytes in size.

[0003] (Field of the Invention) 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 adenoviral (ChAd) vectors and / or SAM vectors.

Background Art

[0004] Human immunodeficiency virus type 1 (HIV-1) infection contributes to global morbidity and mortality, with 38 million people worldwide and 1.1 million people in the United States (US) living with HIV (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 with HIV (PWH). However, discontinuation of ART causes a rapid rebound of viral replication. Therefore, PWH must continue ART throughout their lifespan. The rebound in viremia is due to the persistent reservoir of HIV-infected cells that carry full-length replication-competent proviral HIV DNA not eliminated by ART. The HIV reservoir is established very early in HIV infection, and a substantial portion of the reservoir is transcriptionally silent (i.e., latent) at any given time. Therefore, the latent HIV reservoir cannot be recognized and eliminated by the immune system. Latent HIV proviruses 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 most likely to result from stochastic activation 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 novel therapies that can target and destroy cells latently infected with HIV, which is a major barrier to HIV cure, reduce the HIV reservoir, and ultimately eliminate it, leading to long-term viral remission.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Joint United Nations Programme on HIV / AIDS (UNAIDS). UNAIDS Data. Geneva, Switzerland. 2020 [Non-Patent Document 2] Sengupta and Siliciano, Targeting the Latent Reservoir for HIV-1. Immunity 2018;48(5):872-95 [Summary of the Invention]

[0006] A polynucleotide comprising any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526 is disclosed herein. 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 over the full length of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 to 526, is disclosed herein.

[0007] A polynucleotide comprising an HIV immunogen nucleic acid sequence encoding any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 527 to 528 is disclosed herein.

[0008] 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 the nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogen amino acid sequences of SEQ ID NOs: 527 - 528, wherein the percent identity is over the full length of the nucleic acid sequence of a polypeptide encoding any one of the HIV immunogen amino acid sequences of SEQ ID NOs: 527 - 528, is disclosed herein.

[0009] In some embodiments, the polynucleotide comprises cDNA or mRNA.

[0010] Self - amplifying RNA (SAM or saRNA) comprising any one of the polynucleotides disclosed herein is disclosed herein.

[0011] In some embodiments, the SAM comprises a polynucleotide comprising any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 524 - 526.

[0012] 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 - 526, wherein the percent identity is over the full length of the HIV immunogen nucleic acid sequence.

[0013] In some embodiments, the SAM comprises a polynucleotide comprising an HIV immunogen nucleic acid sequence encoding any one of the HIV immunogen nucleic acid sequences of SEQ ID NOs: 527 - 528.

[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 the nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is over the full length of the nucleic acid sequence of a polypeptide encoding any one of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528.

[0015] In some embodiments, the SAM is of viral origin.

[0016] In some embodiments, the virus is an RNA virus.

[0017] In some embodiments, the RNA virus is a positive-strand single-stranded RNA virus.

[0018] In some embodiments, the virus is selected from alphavirus, flavivirus, nidovirus, nodamura virus, and picornavirus.

[0019] In some embodiments, the alphavirus is selected from Old World (OW) alphaviruses and New World (NW) alphaviruses.

[0020] In some embodiments, the OW alphavirus is selected from Chikunguyna virus (CHIKV), Ross River virus (RRV), Semliki Forest virus (SFV), and Sindbis virus (SINV).

[0021] 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).

[0022] In some embodiments, the SAM is derived from Venezuelan equine encephalitis virus (VEEV).

[0023] In some embodiments, the virus comprises a modified virus genome.

[0024] In some embodiments, the modified virus genome comprises a deletion of one or more genes encoding one or more viral structural proteins.

[0025] In some embodiments, the modified virus genome is produced by replacing one or more viral structural proteins with any of the polynucleotides disclosed herein.

[0026] In some embodiments, the modified virus genome is produced by replacing one or more viral structural proteins with a polynucleotide comprising the nucleic acid sequences of SEQ ID NOs: 524 - 526.

[0027] In some embodiments, the modified virus genome is produced by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence of the nucleic acid sequences of SEQ ID NOs: 524 - 526.

[0028] In some embodiments, the modified viral genome is produced 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 the nucleic acid sequence encoding any of the polypeptides of SEQ ID NOs: 524-526, wherein the percent identity is over the full length of the polypeptides of SEQ ID NOs: 524-526.

[0029] In some embodiments, the modified viral genome is produced by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding any of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0030] In some embodiments, the modified viral genome is produced by replacing one or more viral structural proteins with a polynucleotide encoding any of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0031] In some embodiments, the modified viral genome is produced by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding any of the polypeptides 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 produced by replacing a polynucleotide encoding 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: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of the polypeptides 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 produced by replacing one or more viral structural proteins with a polynucleotide encoding any one of the polypeptides of SEQ ID NOs: 1-344.

[0034] In some embodiments, the modified viral genome is produced by replacing one or more viral structural proteins with a polynucleotide comprising a codon-optimized nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 1-344.

[0035] In some embodiments, the modified viral genome is produced 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 is over the full length of the polypeptides of SEQ ID NOs: 1-344.

[0036] In some embodiments, the virus is replication-deficient.

[0037] Furthermore, self-amplifying RNA (SAM or saRNA) containing a polynucleotide, wherein the polynucleotide comprises any one of the nucleic acid sequences of SEQ ID NOs: 522 to 523, is disclosed herein.

[0038] Furthermore, self-amplifying RNA (SAM or saRNA) containing a 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 of SEQ ID NOs: 522 to 523, and the percent identity is over the full length of the nucleic acid sequences of SEQ ID NOs: 522 to 523, is disclosed herein.

[0039] Furthermore, self-amplifying RNA (SAM or saRNA) containing a polynucleotide, wherein the polynucleotide comprises any one of the nucleic acid sequences of SEQ ID NOs: 524 to 526, is disclosed herein.

[0040] Furthermore, self-amplifying RNA (SAM or saRNA) containing a 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 of SEQ ID NOs: 524 to 526, and the percent identity is over the full length of the nucleic acid sequences of SEQ ID NOs: 524 to 526, is disclosed herein.

[0041] Furthermore, self-amplifying RNA (SAM or saRNA) containing a polynucleotide, wherein the polynucleotide encodes any one of the amino acid sequences of SEQ ID NOs: 527 to 528, is disclosed herein.

[0042] Furthermore, a self-amplifying RNA (SAM or saRNA) comprising 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 is over the entire length of the amino acid sequences of SEQ ID NOs: 527-528, is disclosed herein.

[0043] Furthermore, a self-amplifying RNA (SAM or saRNA) comprising: (a) a first polynucleotide sequence comprising nucleotides 1-7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537-7570 of SEQ ID NO: 522; (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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the entire length of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667-10952 of SEQ ID NO: 522; and (f) a poly-A region, is disclosed herein.

[0044] Furthermore, there is provided 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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 344, and the percent identity is over the full length of SEQ ID NOs: 1 to 344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region. The self-amplifying RNA (SAM or saRNA) is disclosed herein.

[0045] In some embodiments, the third polynucleotide sequence encodes two or more polypeptide segments.

[0046] 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 segments selected from SEQ ID NOs: 1 to 344.

[0047] In some embodiments, one or more of the polypeptide segments are in contact with or fused to an adjacent polypeptide segment.

[0048] In some embodiments, one or more of the polypeptide segments are joined to an adjacent polypeptide segment by one or more peptide linkers.

[0049] In some embodiments, one or more peptide linkers are selected from one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linking sequence, and combinations thereof.

[0050] In some embodiments, a 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).

[0051] In some embodiments, a flexible linker or a polyglycine linker comprises or consists of GG, GGS, GSG, or GGGS (SEQ ID NO: 421).

[0052] In some embodiments, a cleavable linker is selected from 2A cleavable peptides (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A)), furin recognition / cleavage sequences (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), Nef linking sequences, and combinations, derivatives, or variants thereof.

[0053] In some embodiments, a 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).

[0054] 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).

[0055] In some embodiments, the cleavable linker comprises, or consists of, the amino acid sequence of 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).

[0056] 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), and the percent identity is over the full length of SEQ ID NOs: 389 - 391.

[0057] In some embodiments, the Nef linker sequence 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).

[0058] In some embodiments, the promoter sequence comprises the polynucleotide sequence of SEQ ID NO: 529.

[0059] Further disclosed herein is an expression cassette comprising any one of the polypeptides or SAMs disclosed herein operably linked to one or more regulatory sequences.

[0060] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NOs: 524-526 operably linked to one or more regulatory sequences.

[0061] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of the nucleic acid sequences of SEQ ID NOs: 524-526 operably linked to one or more regulatory sequences.

[0062] 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 any one of the polypeptides of SEQ ID NOs: 524-526, wherein the percent identity is over the full length of the polypeptides of SEQ ID NOs: 524-526, and the polynucleotide is 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 encoding any one of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528 operably linked to one or more regulatory sequences.

[0064] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528 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 any of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, operably linked to one or more regulatory sequences.

[0066] 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 any of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of the polypeptides of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, and the polynucleotide is operably linked to one or more regulatory sequences.

[0067] In some embodiments, the expression cassette comprises a polynucleotide encoding any of the polypeptides of SEQ ID NOs: 1-344, operably linked to one or more regulatory sequences.

[0068] In some embodiments, the expression cassette comprises a polynucleotide comprising a codon-optimized nucleic acid sequence encoding any of the polypeptides of SEQ ID NOs: 1-344, operably linked to one or more regulatory sequences.

[0069] 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 the nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 1-344, wherein the percent identity is over the full length of the polypeptides of 1-344, and the polynucleotide is operably linked to one or more regulatory sequences.

[0070] In some embodiments, the polynucleotide is operably linked to and under the control of a constitutive promoter.

[0071] In some embodiments, the promoter is selected from the CMV promoter, CAG promoter, EF1a promoter, and 26S subgenomic promoter.

[0072] In some embodiments, the promoter comprises any one of the promoter sequences of SEQ ID NOs: 529-530.

[0073] Further disclosed herein are lipid nanoparticles (LNPs) comprising any one of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0074] In some embodiments, the LNP comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0075] In some embodiments, the LNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0076] 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 522-523, a polynucleotide, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0077] In some embodiments, the LNP comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0078] In some embodiments, the LNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0079] 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 524-526, a polynucleotide, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0080] In some embodiments, the LNP comprises a polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 527-528, 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 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0082] In some embodiments, the LNP comprises a SAM, wherein the SAM comprises: (a) a first polynucleotide sequence comprising nucleotides 1-7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537-7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667-10952 of SEQ ID NO: 522; and (f) a polyA region.

[0083] In some embodiments, the LNP comprises a SAM, wherein the SAM 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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 344, and the percent identity is over the full length of SEQ ID NOs: 1 to 344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0084] In some embodiments, the LNP comprises or is synthesized from an ionizable lipid, a phospholipid, cholesterol, a PEGylated lipid, or any combination thereof.

[0085] In some embodiments, the LNP comprises or is synthesized from the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0086] Further disclosed herein are polymeric nanoparticles (PNP) comprising any of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0087] In some embodiments, the PNP comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 522 to 523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0088] In some embodiments, the PNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0089] In some embodiments, the PNP is 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 over the full length of the nucleic acid sequences 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0091] In some embodiments, the PNP comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0092] In some embodiments, the PNP is 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 over the full length of the nucleic acid sequences 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 nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0094] In some embodiments, the PNP is 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0095] In some embodiments, the PNP comprises a SAM, wherein the SAM comprises: (a) a first polynucleotide sequence comprising nucleotides 1-7512 of SEQ ID NO: 522; (b) a promoter sequence; (c) a second polynucleotide sequence comprising nucleotides 7537-7570 of SEQ ID NO: 522; (d) a third polynucleotide sequence encoding a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667-10952 of SEQ ID NO: 522; and (f) a polyA region.

[0096] In some embodiments, the PNP comprises a SAM, and the SAM 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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 344, and the percent identity is over the full length of SEQ ID NOs: 1 to 344, the third polynucleotide sequence, (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522, and (f) a polyA region.

[0097] In some embodiments, 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.

[0098] 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), an oltenin-derived dendrimer, or any combination thereof.

[0099] In some embodiments, the PNP comprises or is synthesized from a mannosylated PEI polyplex.

[0100] In some embodiments, the PNP comprises or is synthesized from a bioreducible linear cationic polymer (pABOL).

[0101] Furthermore, disclosed herein are nanoemulsions comprising any one of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0102] In some embodiments, the nanoemulsion comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0103] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 522-523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0104] 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 over the full length of the nucleic acid sequences 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0106] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 524 to 526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0107] In some embodiments, the nanoemulsion is 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 to 526, wherein the percent identity is over the full length of the nucleic acid sequences of SEQ ID NOs: 524 to 526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0108] In some embodiments, the nanoemulsion comprises a polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 527 to 528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0109] In some embodiments, the nanoemulsion is 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 to 528, wherein the percent identity is over the full length of the nucleic acid sequences of SEQ ID NOs: 527 to 528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0110] In some embodiments, the nanoemulsion comprises SAM, and the SAM comprises (a) a first polynucleotide sequence comprising nucleotides 1 to 7,512 of SEQ ID NO: 522, (b) a promoter sequence, (c) a second polynucleotide sequence comprising nucleotides 7,537 to 7,570 of SEQ ID NO: 522, (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 any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, (e) a fourth polynucleotide sequence comprising nucleotides 10,667 to 10,952 of SEQ ID NO: 522, and (f) a polyA region.

[0111] In some embodiments, the nanoemulsion comprises SAM, and the SAM comprises (a) a first polynucleotide sequence comprising nucleotides 1 to 7,512 of SEQ ID NO: 522, (b) a promoter sequence, (c) a second polynucleotide sequence comprising nucleotides 7,537 to 7,570 of SEQ ID NO: 522, (d) a third polynucleotide sequence encoding one or more polypeptide segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 - 344, and the percent identity is over the full length of SEQ ID NOs: 1 - 344, (e) a fourth polynucleotide sequence comprising nucleotides 10,667 to 10,952 of SEQ ID NO: 522, and (f) a polyA region.

[0112] In some embodiments, the nanoemulsion is a water-in-oil emulsion.

[0113] In some embodiments, the nanoemulsion comprises squalene, sorbitan trioleate, polysorbate 80, DOTAP, or any combination thereof.

[0114] Furthermore, an expression vector is disclosed herein that comprises any one of the polynucleotides, SAMs, or expression cassettes disclosed herein.

[0115] In some embodiments, the expression vector comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 522 - 523, or a SAM or expression cassette comprising such a polynucleotide.

[0116] In some embodiments, the expression vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 522 - 523, or a SAM or expression cassette comprising such a polynucleotide.

[0117] 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 any one of the nucleic acid sequences of SEQ ID NOs: 522 - 523, wherein the percent identity is over the entire length of the nucleic acid sequences 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 any one of the nucleic acid sequences of SEQ ID NOs: 524 - 526, or a SAM or expression cassette comprising such a polynucleotide.

[0119] In some embodiments, the expression vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 524 to 526, or a SAM or an expression cassette comprising such a polynucleotide.

[0120] 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 any one of the nucleic acid sequences of SEQ ID NOs: 524 to 526, wherein the percent identity is over the full length of the nucleic acid sequence of SEQ ID NOs: 524 to 526, or a SAM or an expression cassette comprising such a polynucleotide.

[0121] In some embodiments, the expression cassette comprises a polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 527 to 528, or a SAM or an expression cassette comprising such a polynucleotide.

[0122] In some embodiments, the expression vector 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 to 528, wherein the percent identity is over the full length of the nucleic acid sequence of SEQ ID NOs: 527 to 528, or a SAM or an expression cassette comprising such a polynucleotide.

[0123] In some embodiments, the expression vector comprises SAM, and SAM 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 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 any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0124] In some embodiments, the expression vector comprises SAM, and SAM 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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 - 344, and the percent identity is over the full 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.

[0125] In some embodiments, the expression vector is a plasmid vector, a bacterial vector, or a viral expression vector.

[0126] In some embodiments, the expression vector is a viral expression vector.

[0127] In some embodiments, the expression vector is a DNA virus or an RNA virus.

[0128] In some embodiments, the viral expression vector is replication-deficient, replication-deleted, replication-attenuated, or replication-competent.

[0129] In some embodiments, the viral expression vector is derived from a virus selected from adenovirus, adeno-associated virus, arenavirus, alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, maraba virus, and vaccinia virus.

[0130] In some embodiments, the viral expression vector is derived from a virus from a taxonomic family selected from Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia 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).

[0131] In some embodiments, the viral expression vector is an arenavirus selected from Lymphocytic choriomeningitis mammarenavirus (LCMV), Calomyscallymphocytic choriomeningitis mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

[0132] In some embodiments, the viral expression vector is an arenavirus vector selected from Lymphocytic choriomeningitis mammarenavirus (LCMV), or Calomyscallymphocytic choriomeningitis mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus).

[0133] In some embodiments, the arenavirus vector comprises a two-segmented genome.

[0134] In some embodiments, the arenavirus vector comprises a three-segmented genome.

[0135] In some embodiments, the viral expression vector is a human adenovirus or a simian adenovirus.

[0136] In some embodiments, the simian adenovirus is selected from chimpanzee adenovirus, gorilla adenovirus, and rhesus adenovirus.

[0137] In some embodiments, the viral expression vector is an adenovirus selected from 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 adenovirus.

[0138] In some embodiments, the ChAd is selected from 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), 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).

[0139] In some embodiments, the ChAd is ChAd68.

[0140] In some embodiments, the gorilla adenovirus is selected from GC44, GC45, and GC46.

[0141] 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.

[0142] In some embodiments, the human adenovirus or simian adenovirus comprises a modification of one or more adenovirus genes.

[0143] In some embodiments, the one or more adenovirus genes are selected from E1, E3, and E4ORF2-4.

[0144] In some embodiments, the modification comprises a deletion, substitution, or insertion of one or more nucleotides into one or more adenovirus genes.

[0145] In some embodiments, the human adenovirus or simian adenovirus comprises a deletion of at least a portion of the E1 region.

[0146] In some embodiments, the polynucleotide is inserted into a deletion of at least a portion of the E1 region.

[0147] In some embodiments, the expression vector comprises any one of the nucleic acid sequences of SEQ ID NOs: 520-521.

[0148] 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 520-521.

[0149] Furthermore, an expression vector containing any one of the nucleic acid sequences of SEQ ID NOs: 524 to 526 is disclosed herein.

[0150] Furthermore, 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 to 526, wherein the percent identity is over the full length of the nucleic acid sequences of SEQ ID NOs: 524 to 526, is disclosed herein.

[0151] Furthermore, an expression vector containing a polynucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 527 to 528 is disclosed herein.

[0152] Furthermore, 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 to 528, wherein the percent identity is over the full length of the amino acid sequences of SEQ ID NOs: 527 to 528, is disclosed herein.

[0153] Furthermore, an expression vector is disclosed herein, which comprises: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (b) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (c) a third polynucleotide sequence encoding a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 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.

[0154] Furthermore, an expression vector is disclosed herein, which comprises: (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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 344, and the percent identity is over the full length of SEQ ID NOs: 1 to 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.

[0155] In some embodiments, the third polynucleotide sequence encodes two or more polypeptide segments.

[0156] 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 segments selected from SEQ ID NOs: 1-344.

[0157] In some embodiments, one or more of the polypeptide segments contact or are fused to an adjacent polypeptide segment.

[0158] In some embodiments, one or more of the polypeptide segments are joined to an adjacent polypeptide segment by one or more peptide linkers.

[0159] In some embodiments, one or more of the peptide linkers are selected from one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef linker sequence, and combinations thereof.

[0160] 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).

[0161] In some embodiments, the flexible linker or polyglycine linker comprises or consists of GG, GGS, GSG, or GGGS (SEQ ID NO: 421).

[0162] In some embodiments, the cleavable linker is selected from 2A cleavable peptides (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A)), furin recognition / cleavage sequences (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)), Nef linker sequences, and combinations, derivatives, or variants thereof.

[0163] 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).

[0164] 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), and the percent identity is over the full length of SEQ ID NOS: 384 - 388.

[0165] In some embodiments, the cleavable linker comprises or consists of the amino acid sequence of 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).

[0166] 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), and the percent identity is over the full length of SEQ ID NOs: 389 - 391.

[0167] In some embodiments, the Nef linker sequence 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).

[0168] In some embodiments, the promoter sequence comprises the polynucleotide sequence of SEQ ID NO: 529.

[0169] Further disclosed herein is a viral vector comprising any one of the polynucleotides, SAMs, expression cassettes, or expression vectors disclosed herein.

[0170] In some embodiments, the viral vector comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 522 - 523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0171] In some embodiments, the viral vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 522 - 523, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0172] 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 over the entire length of the nucleic acid sequences of SEQ ID NOs: 522-523, a polynucleotide, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0173] In some embodiments, the viral vector comprises a polynucleotide comprising any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0174] In some embodiments, the viral vector comprises a polynucleotide comprising a codon-optimized nucleic acid sequence of any one of the nucleic acid sequences of SEQ ID NOs: 524-526, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0175] 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 over the entire length of the nucleic acid sequences of SEQ ID NOs: 524-526, a polynucleotide, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0176] In some embodiments, the viral vector comprises a polynucleotide comprising a nucleic acid sequence encoding any one of the polypeptides of SEQ ID NOs: 527-528, 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 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 527-528, or a SAM, expression cassette, or expression vector comprising such a polynucleotide.

[0178] In some embodiments, the viral vector comprises a SAM, wherein the SAM comprises (a) a first polynucleotide sequence comprising nucleotides 1-7512 of SEQ ID NO: 522, (b) a promoter sequence, (c) a second polynucleotide sequence comprising nucleotides 7537-7570 of SEQ ID NO: 522, (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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, (e) a fourth polynucleotide sequence comprising nucleotides 10667-10952 of SEQ ID NO: 522, and (f) a poly A region.

[0179] In some embodiments, the viral vector comprises a SAM, wherein the SAM 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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 344, and the percent identity is over the full length of SEQ ID NOs: 1 to 344; (e) a fourth polynucleotide sequence comprising nucleotides 10667 to 10952 of SEQ ID NO: 522; and (f) a polyA region.

[0180] In some embodiments, the viral vector is derived from or based on a DNA virus or an RNA virus.

[0181] In some embodiments, the viral vector is replication-deficient, replication-deleted, replication-attenuated, or replication-competent.

[0182] In some embodiments, the viral expression vector is derived 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.

[0183] In some embodiments, the viral vector is derived from or based on a virus from a taxonomic family selected from Adenoviridae, Arenaviridae, Herpesviridae (e.g., cytomegalovirus), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia 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).

[0184] In some embodiments, the viral vector is derived from or based on an arenavirus selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Calomys callosus arenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Lujo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV).

[0185] In some embodiments, the viral vector is derived from or based on an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Calomys callosus arenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus).

[0186] In some embodiments, the arenavirus vector comprises a two-segmented genome.

[0187] In some embodiments, the arenavirus vector comprises a three-segmented genome.

[0188] In some embodiments, the viral vector is a human adenovirus or a simian adenovirus.

[0189] In some embodiments, the simian adenovirus is selected from chimpanzee adenovirus, gorilla adenovirus, and rhesus adenovirus.

[0190] In some embodiments, the viral vector is derived from or based on an adenovirus selected from 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 adenovirus.

[0191] In some embodiments, the ChAd is selected from 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), 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).

[0192] In some embodiments, the ChAd is ChAd68.

[0193] In some embodiments, the gorilla adenovirus is selected from GC44, GC45, and GC46.

[0194] 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.

[0195] In some embodiments, the human adenovirus or simian adenovirus comprises a modification of one or more adenoviral genes.

[0196] In some embodiments, the one or more adenoviral genes are selected from E1, E3, and E4ORF2-4.

[0197] In some embodiments, the modification comprises a deletion, substitution, or insertion of one or more nucleotides into one or more adenoviral genes.

[0198] In some embodiments, the human adenovirus or simian adenovirus comprises a deletion of at least a portion of the E1 region.

[0199] In some embodiments, the polynucleotide is inserted into a deletion of at least a portion of the E1 region.

[0200] In some embodiments, the viral vector comprises any one of the nucleic acid sequences of SEQ ID NOs: 520-521.

[0201] 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 over the full length of the nucleic acid sequences of SEQ ID NOs: 520-521.

[0202] In some embodiments, the viral vector comprises an expression vector, and the expression vector comprises: (a) a first polynucleotide sequence comprising nucleotides 1 to 593 of SEQ ID NO: 520; (b) a promoter sequence; (b) a second polynucleotide sequence comprising nucleotides 1196 to 1255 of SEQ ID NO: 520; (c) 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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, wherein the percent identity is over the full length of SEQ ID NOs: 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.

[0203] In some embodiments, the viral vector comprises an expression vector, and the expression vector comprises: (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 segments, wherein the one or more polypeptide segments comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1-344, and the percent identity is over the full 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.

[0204] Furthermore, host cells are disclosed herein that contain any of the polynucleotides, SAM, expression cassettes, expression vectors, viral vectors, LNPs, PNP, or nanoemulsions disclosed herein.

[0205] In some embodiments, one or more polynucleotides are not integrated into the host cell genome and are, for example, episomal.

[0206] In some embodiments, one or more polynucleotides are integrated into the host cell genome.

[0207] In some embodiments, the host cell is a mammalian cell.

[0208] In some embodiments, the mammalian cell is a human cell.

[0209] In some embodiments, the mammalian cell is not a human cell.

[0210] In some embodiments, the host cell is in vitro.

[0211] In some embodiments, the host cell is in vivo.

[0212] Furthermore, polypeptides are disclosed herein that contain any of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528.

[0213] Furthermore, polypeptides are disclosed herein that contain 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 of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is over the full length of the HIV immunogen amino acid sequences of SEQ ID NOs: 527-528.

[0214] Furthermore, a composition is disclosed herein comprising (a) one or more of the polynucleotides, SAMs, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein, and (b) a pharmaceutically acceptable diluent, carrier, or excipient.

[0215] In some embodiments, the composition comprises two or more of the polynucleotides, SAMs, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0216] Furthermore, a composition is disclosed herein comprising (a) one or more of the SAMs, expression cassettes, LNPs, PNPs, or nanoemulsions disclosed herein, and (b) one or more of the expression cassettes or viral vectors disclosed herein.

[0217] Furthermore, a composition is disclosed herein comprising (a) one or more of the polypeptides disclosed herein, and (b) a pharmaceutically acceptable diluent, carrier, or excipient.

[0218] In some embodiments, the composition comprises two or more of the polypeptides disclosed herein.

[0219] In some embodiments, any of the compositions disclosed herein further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

[0220] In some embodiments, any of the compositions disclosed herein further comprises one or more of an adjuvant, an immunostimulant, a detergent, a micelle-forming agent, and an oil.

[0221] In some embodiments, the immunostimulant 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.

[0222] In some embodiments, cytokines 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.

[0223] In some embodiments, the non-coding immunostimulatory polynucleotide is selected from pathogen-activated molecular pattern (PAMP), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides, and immunostimulatory RNA (isRNA, e.g., CV8102).

[0224] In some embodiments, the composition is formulated for administration via a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., intraoral, intranasal, rectal, vaginal).

[0225] In some embodiments, the composition is formulated as a liquid.

[0226] In some embodiments, the composition is lyophilized.

[0227] Further disclosed herein is a kit comprising one or more components selected from one or more of the polynucleotides, SAMs, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, nanoemulsions, or polypeptides disclosed herein.

[0228] Further disclosed herein is a kit comprising (a) a first component selected from any of the SAMs, LNPs, PNPs, or nanoemulsions disclosed herein, and (b) a second component comprising any of the expression vectors or viral vectors disclosed herein.

[0229] Furthermore, kits are disclosed herein that include one or more unit doses of any of the polynucleotides, SAMs, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, nanoemulsions, or polypeptides disclosed herein.

[0230] Furthermore, kits are disclosed herein that include (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.

[0231] Furthermore, kits are disclosed herein that include one or more unit doses of any of the polypeptides disclosed herein.

[0232] In some embodiments, one or more components are in a single container.

[0233] In some embodiments, one or more components are in two or more separate containers.

[0234] In some embodiments, the first and second components are in a single container.

[0235] In some embodiments, the first and second components are in two or more separate containers.

[0236] In some embodiments, one or more unit doses are in a single container.

[0237] In some embodiments, one or more unit doses are in two or more separate containers.

[0238] In some embodiments, one or more containers are selected from the group consisting of vials, ampoules, and pre-filled syringes.

[0239] In some embodiments, one or more containers contain one or more SAMs, one or more LNPs, one or more PNP, one or more nanoemulsions, one or more vectors, or one or more polypeptides in an aqueous solution.

[0240] In some embodiments, the one or more unit doses are the same.

[0241] In some embodiments, the one or more unit doses are different.

[0242] In some embodiments, the unit dose of one or more viral vectors is from about 10 3 to about 10 15 in the range of viral focus forming units (FFU), plaque forming units (PFU), infectious units (IU), or viral particles (vp).

[0243] In some embodiments, the unit dose of one or more viral vectors is from about 10 4 to about 10 7 in viral FFU, PFU, IU, or vp.

[0244] In some embodiments, the unit dose of one or more viral vectors is from 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 in viral FFU, PFU, IU, or vp.

[0245] In some embodiments, the unit dose of one or more SAMs is in the range of about 1 μg to about 1000 μg.

[0246] In some embodiments, the unit dose of one or more SAMs is from about 15 μg to about 500 μg.

[0247] In some embodiments, the unit dose of one or more SAMs is from about 1 μg to about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg.

[0248] 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.

[0249] 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 inhibitors of CD47, one or more immune-based therapies, one or more broadly neutralizing antibodies (bnAbs), and one or more antiviral agents.

[0250] In some embodiments, the one or more agents that activate latent HIV are one or more latency reversing agents (LRAs).

[0251] In some embodiments, one or more LRAs are selected from the group consisting 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 agonists or activators of second mitochondria-derived activator of caspase (SMAC) mimetics.

[0252] In some embodiments, the TLR agonist or activator is selected from the group consisting of TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists.

[0253] In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620 (vesatolimod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod), and / or 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).

[0254] In some embodiments, any of the compositions or kits disclosed herein further comprises GS 9620 (vesatolimod).

[0255] In some embodiments, the TLR9 agonist is selected from the group consisting of AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, retunimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatrimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, refitrimod (MGN-1703), CYT-003, CYT-003-QbG10, chilothrimod, and PUL-042.

[0256] In some embodiments, any of the compositions or kits disclosed herein further comprises refitrimod (MGN-1703).

[0257] 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.

[0258] In some embodiments, one or more cytokines 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.

[0259] In some embodiments, the receptor agonist is an agonist of one or more receptors selected from fms related tyrosine kinase 3 (FLT3), stimulator of interferon gene (STING) receptor, DExD / H-box helicase 58 (DDX58, also known as RIG-I), nucleotide binding oligomerization domain containing 2 (NOD2).

[0260] In some embodiments, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors are 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 immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, 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 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, one Ig domain, and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR3DL1), and inhibit checkpoint proteins or receptors selected from the group consisting of.

[0261] In some embodiments, one or more agonists, activators or stimulators of a T cell-stimulating immune checkpoint protein or receptor agonize, activate or stimulate a T cell-stimulating immune checkpoint protein or receptor selected from the group consisting of CD27, CD70; CD40, CD40LG; inducible T cell co-stimulatory molecule (ICOS, CD278); inducible T cell co-stimulatory molecule 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).

[0262] In some embodiments, inhibitors of CTLA4 are selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), 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.

[0263] In some embodiments, the inhibitor of PD-L1 (CD274) or PD-1 (PDCD1) is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pidilizumab, AB122 (zimberelimab), 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 (toripalimab), JNJ-63723283, genolimzumab (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 (dostarlimab), 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.

[0264] In some embodiments, one or more antiviral agents are selected from the group consisting of an HIV protease inhibitor, an HIV reverse transcriptase inhibitor, an HIV integrase inhibitor, an HIV non-catalytic site (or allosteric) integrase inhibitor, an HIV entry (fusion) inhibitor, an HIV maturation inhibitor, and a capsid inhibitor.

[0265] Further disclosed herein is a method for doing so in a subject in need of inducing an immune response against human immunodeficiency virus (HIV). In some embodiments, the method comprises administering to the subject any one or more of the polynucleotides, SAM, LNP, PNP, nanoemulsion, expression cassette, expression vector, viral vector, or polypeptide disclosed herein, or any combination thereof.

[0266] Also disclosed herein is a method for doing so in a subject in need of treating or preventing human immunodeficiency virus (HIV). In some embodiments, the method comprises administering to the subject any one or more of the polynucleotides, SAM, LNP, PNP, nanoemulsion, expression cassette, expression vector, viral vector, or polypeptide disclosed herein, or any combination thereof.

[0267] In some embodiments, the subject is infected with HIV-1, suspected of being infected with HIV-1, or at risk of infection with HIV-1.

[0268] In some embodiments, the subject is chronically infected with HIV-1.

[0269] In some embodiments, the subject is acutely infected with HIV-1.

[0270] In some embodiments, the subject has HIV-1 infection at Fiebig stage IV or earlier, such as, for example, Fiebig stage III, Fiebig stage II, or Fiebig stage I.

[0271] In some embodiments, administering comprises an administration route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., intraoral, intranasal, rectal, vaginal).

[0272] In some embodiments, the method comprises administering one or more viral vectors in a dose range of about 10 3 ~ about 10 15 viral focus forming units (FFU) or plaque forming units (PFU) or infectious units (IU) or viral particles (vp).

[0273] In some embodiments, the one or more viral vectors are administered in a dose range of about 10 4 ~ about 10 7 viral FFU or PFU or IU or vp, such as, 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 viral FFU or PFU or IU or vp.

[0274] In some embodiments, the method comprises administering one or more of the SAM, LNP, PNP, or nanoemulsions disclosed herein in a dose range of about 1 μg to about 1000 μg.

[0275] In some embodiments, the one or more SAM, LNP, PNP, or nanoemulsions are administered in a dose range of about 15 μg to about 500 μg.

[0276] In some embodiments, one or more SAMs, LNPs, PNPs, or nanoemulsions are administered in a dosage range of about 1 μg to about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg.

[0277] In some embodiments, any of the methods disclosed herein include a prime-boost regimen, where the prime-boost regimen includes (a) administering a priming composition at a first time point and 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 and a boosting composition at a second time point, one or more repetitions (e.g., prime-boost-prime-boost, etc.), or (c) administering a priming composition at a first time point, one or more boosting compositions at one or more subsequent time points, and a priming composition at a subsequent time point after the administration of the boosting composition, one or more repetitions (e.g., prime-boost-boost-prime, etc.).

[0278] In some embodiments, the administration of the priming composition and one or more boosting compositions is spaced at intervals of at least one week, two weeks, three weeks, or one month, e.g., at intervals of at least two, three, four, five, or six months.

[0279] In some embodiments, the priming composition and the one or more boosting compositions include polynucleotides encoding the same immunogenic polypeptide.

[0280] In some embodiments, the priming composition and the boosting composition include polynucleotides encoding different immunogenic polypeptides.

[0281] 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.

[0282] In some embodiments, the priming composition and the boosting composition comprise different polypeptides, polynucleotides, SAMs, LNPs, PNPs, nanoemulsions, or viral vectors.

[0283] In some embodiments, any of the methods disclosed herein comprise priming with a first polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector, and boosting with a second polynucleotide, SAM, LNP, PNP, nanoemulsion, or viral vector.

[0284] In some embodiments, the prime-boost regimen comprises: (i) priming with a viral expression vector and 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 boosting with a viral expression vector, (iii) priming with a first viral expression vector and boosting with a second viral expression vector (wherein the first and second viral expression vectors are from the same, related, or unrelated taxonomic families), (iv) priming with a first replication-deficient viral expression vector and boosting with a second replication-deficient viral expression vector (wherein the first and second replication-deficient viral expression vectors are from the same, related, or unrelated taxonomic families), (v) priming with a first attenuated replication-deficient viral expression vector and boosting with a second replication-attenuated replication-deficient viral expression vector (wherein the first and second replication-attenuated viral expression vectors are from the same, related, or unrelated taxonomic families), (vi) priming with a replication-deficient viral expression vector and boosting with a replication-attenuated viral expression vector, (vii) priming with a replication-attenuated viral expression vector and boosting with a replication-deficient viral expression vector, (viii) priming with a viral vector and boosting with a composition comprising SAM, (ix) priming with an adenovirus expression vector and boosting with a polynucleotide (wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA), (x) priming with an adenovirus vector and boosting with a composition comprising SAM, (xi) priming with a ChAd expression vector and boosting with a polynucleotide (wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA), or (xii) priming with a ChAd vector and boosting with a composition comprising SAM.

[0285] In some embodiments, the subject has not received antiretroviral therapy (ART), or ART is discontinued prior to administration of one or more polynucleotides, polypeptides, SAM, LNPs, PNPs, nanoemulsions, expression cassettes, or viral vectors.

[0286] In some embodiments, ART is discontinued after one or more administrations of a polynucleotide, polypeptide, SAM, LNP, PNP, nanoemulsion, expression cassette, or viral vector.

[0287] In some embodiments, any of the methods disclosed herein further comprises administering to the subject one or more additional therapeutic agents, such as two, three, four, or more additional therapeutic agents.

[0288] In some embodiments, the one or more 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 antiviral agents, one or more immune-based therapies, and one or more broadly neutralizing antibodies (bnAbs).

[0289] In some embodiments, the one or more agents that activate latent HIV are one or more latency reversing agents (LRAs).

[0290] In some embodiments, one or more LRAs are selected from 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, apoptosis protein inhibitor (IAP) antagonists, and agonists or activators of second mitochondria-derived caspase activator (SMAC) mimetics.

[0291] In some embodiments, one or more TLR agonists or activators are selected from the group consisting of TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists.

[0292] In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620 (vesatolimod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod), and / or 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).

[0293] In some embodiments, any of the methods disclosed herein further comprises administering GS 9620 (vesatolimod) to a subject.

[0294] In some embodiments, the TLR9 agonist is selected from the group consisting of AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, retenimod, 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.

[0295] In some embodiments, any of the methods disclosed herein further comprises administering refitrimod (MGN-1703).

[0296] In some embodiments, one or more bnAbs are HIV bnAbs.

[0297] In some embodiments, any of the methods disclosed herein further comprises administering one or more bnAbs.

[0298] In some embodiments, the HIV bnAbs are highly effective against most prevalent strains of HIV, neutralize a broad range of genetically diverse HIV-1 subtypes, and / or potently neutralize a significant percentage of primary isolates, or exhibit some ability to reach class 2 and 3 viruses that are more difficult to neutralize across clades.

[0299] In some embodiments, the bnAbs are directed to the surface of a larger site that encompasses the Env: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 the interface between gp41 and gp120.

[0300] 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, CH01, 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, PGT144, PGT145, VRC-CH30, CRC-CH31, CRC-CH32, CRC-CH33, CRC-CH34, VRC-PG04, VRC-PG04b, VRC01, VRC02, VRC03, AND Z13.

[0301] In some embodiments, the bnAb is an anti-CD4 bnAb.

[0302] In some embodiments, the anti-CD4 bnAb is selected from 3BNC117 and VRC01.

[0303] In some embodiments, the bnAb is an anti-V3 bnAb.

[0304] In some embodiments, the anti-V3 bnAb is 10-1074.

[0305] In some embodiments, one or more interleukin receptor agonists are agonists of interleukin selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-a, IFN-g, GM-CSF, FLT3LG.

[0306] In some embodiments, 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.

[0307] In some embodiments, one or more receptor agonists are agonists of receptors selected from 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 2 (NOD2).

[0308] In some embodiments, one or more inhibitors of T cell inhibitory immune checkpoint proteins or receptors are 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 immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, 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 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, one Ig domain, and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR3DL1), an inhibitor of a T cell inhibitory immune checkpoint protein or receptor selected from the group consisting of.

[0309] In some embodiments, one or more agonists, activators, or stimulators of a T cell-stimulating immune checkpoint protein or receptor agonize, activate, or stimulate a T cell-stimulating immune checkpoint protein or receptor selected from CD27, CD70; CD40, CD40LG; inducible T cell co-stimulatory molecule (ICOS, CD278); inducible T cell co-stimulatory molecule 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).

[0310] In some embodiments, inhibitors of CTLA4 are selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), 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.

[0311] In some embodiments, the inhibitor of PD-L1 (CD274) or PD-1 (PDCD1) is selected from the group consisting of pembrolizumab, nivolumab, semiprimab, pidilizumab, AB122 (zinberelimab), 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 (balstilimab), JS-001 (toripalimab), JNJ-63723283, genolimzumab (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 (dostarlimab), 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.

[0312] In some embodiments, one or more antiviral agents are selected from the group consisting of an HIV protease inhibitor, an HIV reverse transcriptase inhibitor, an HIV integrase inhibitor, an HIV non-catalytic site (or allosteric) integrase inhibitor, an HIV entry (fusion) inhibitor, an HIV maturation inhibitor, and a capsid inhibitor.

[0313] In some embodiments, after 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, the subject exhibits no symptoms of HIV or AIDS 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).

[0314] In some embodiments, after 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, the subject has a viral load of less than 500, such as less than 400, less than 300, less than 200, less than 100, less than 50 copies / ml of blood 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).

[0315] 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 inducing an immune response against human immunodeficiency virus (HIV) in a subject in need thereof.

[0316] Further disclosed herein is the use of any one of the polynucleotides, SAM, expression cassettes, LNPs, PNP, nanoemulsions, expression vectors, viral vectors, polypeptides, or compositions disclosed herein for performing the same in a subject in need of treating or preventing human immunodeficiency virus (HIV).

[0317] Further disclosed herein is the use of any one of the polynucleotides, SAM, expression cassettes, LNPs, PNP, nanoemulsions, expression vectors, viral vectors, polypeptides, or compositions disclosed herein as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0318]

Figure 1

[0319]

Figure 2

[0320]

Figure 3

[0321]

Figure 4A

Figure 4B

[0322]

Figure 5A

Figure 5B

Figure 5C

[0323]

Figure 6

[0324]

Figure 7

[0325]

Figure 8

[0326]

Figure 9

[0327]

Figure 10A

Figure 10B

Figure 10C

Figure 10D

[0328]

Figure 11

[0329]

Figure 12

[0330]

Figure 13

[0331]

Figure 14A

[0332]

Figure 14B

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Figure 15

[0334]

Figure 16

[0335]

Figure 17

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Figure 18

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Figure 19

[0338]

Figure 20

DETAILED DESCRIPTION OF THE INVENTION

[0339] An HIV immunogenic polypeptide is provided herein. In some embodiments, the HIV immunogenic polypeptide comprises the amino acid sequences of SEQ ID NOs: 527-528.

[0340] Furthermore, a polynucleotide encoding the HIV immunogenic polypeptide is provided herein. In some embodiments, 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 HIV immunogenic nucleic acid sequences of SEQ ID NOs: 524-526, wherein the percent identity is over the full length of the HIV immunogenic nucleic acid sequences of SEQ ID NOs: 524-526. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any one of the HIV immunogenic nucleic acid sequences of SEQ ID NOs: 527-528. In some embodiments, 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 the nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogenic amino acid sequences of SEQ ID NOs: 527-528, wherein the percent identity is over the full length of the nucleic acid sequence of a polynucleotide encoding any one of the HIV immunogenic amino acid sequences of SEQ ID NOs: 527-528.

[0341] Furthermore, an expression cassette comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein is 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0342] Furthermore, an expression vector comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein is disclosed herein. In some embodiments, the expression vector 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 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: 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 any one of the nucleic acid sequences 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0343] Furthermore, self-amplifying RNA (SAM) comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein is disclosed herein. In some embodiments, the SAM 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 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 nucleic acid sequences 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0344] Furthermore, viral vectors comprising a polynucleotide encoding any of the HIV immunogenic polypeptides disclosed herein are disclosed herein. In some embodiments, the viral vector 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 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: 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 any one of the nucleic acid sequences of SEQ ID NOs: 524-526.

[0345] Furthermore, lipid nanoparticles (LNP), polymer nanoparticles (PNP), or nanoemulsions comprising any of the polynucleotides, expression cassettes, expression vectors, or viral vectors disclosed herein are disclosed herein.

[0346] Furthermore, cells comprising any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNP, PNP, or nanoemulsions disclosed herein are disclosed herein.

[0347] Furthermore, compositions comprising any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNP, PNP, or nanoemulsions disclosed herein are disclosed herein.

[0348] Furthermore, kits are disclosed herein that comprise any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein.

[0349] Furthermore, methods are disclosed herein of using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein to effect the same in a subject in need of inducing an immune response against human immunodeficiency virus (HIV).

[0350] Furthermore, methods are disclosed herein of using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein to effect the same in a subject in need of treating or preventing human immunodeficiency virus (HIV).

[0351] Furthermore, methods are disclosed herein of using any of the polypeptides, polynucleotides, expression cassettes, expression vectors, viral vectors, LNPs, PNPs, or nanoemulsions disclosed herein to effect the same in a subject in need of treating or preventing human immunodeficiency virus (HIV). HIV immunogenic polypeptide

[0352] HIV immunogenic polypeptides are disclosed herein. As used herein, the phrase "HIV immunogenic polypeptide" refers to a polypeptide that can induce 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 induces or is capable of inducing an immune response. The phrases "HIV immunogenic polypeptide" and "fusion polypeptide" may be used interchangeably. Polypeptide segment

[0353] The HIV immunogenic polypeptide comprises a plurality of polypeptides or peptide segments encoded by one or more HIV-1 genes. A "segment" of an HIV immunogenic polypeptide described herein (e.g., a fusion polypeptide) is a continuous sequence of at least 8 amino acids with respect to a reference sequence (e.g., the HIV-1 HXB2 reference sequences 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 in the sense that they are assembled from connected or linked polypeptides or peptide segments of two or more HIV-1 proteins. With respect to the HIV-1 protein reference sequences, a polypeptide or peptide segment can correspond to discontinuous sequences of the same HIV-1 protein or different HIV-1 proteins. Generally, the HIV immunogenic polypeptides (e.g., fusion polypeptides) do not occur naturally and can be produced synthetically or recombinantly.

[0354] Regarding the HIV-1 genes encoding polypeptide segments used to construct the fusion polypeptides described herein, in various embodiments, the fusion polypeptide comprises a plurality of polypeptide segments of one or more (e.g., two or more, three or more, four or more) HIV-1 proteins encoded by one 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 segments consists only of polypeptide segments encoded by the HIV-1 genes Env, Gag, Nef, and Pol, and for example, does not include polypeptide segments encoded by the HIV-1 Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide segments consists only of polypeptide segments encoded by the HIV-1 genes Gag, Nef, and Pol, and for example, does not include polypeptide segments encoded by the HIV-1 Env, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide segments consists only of polypeptide segments encoded by the HIV-1 genes Gag and Nef, and for example, does not include polypeptide segments encoded by the HIV-1 Env, Pol, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide segments consists only of polypeptide segments encoded by the HIV-1 genes Pol and Nef, and for example, does not include polypeptide segments encoded by the HIV-1 Env, Gag, Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide segments consists only of polypeptide segments encoded by the HIV-1 genes Pol and Env, and for example, does not include polypeptide segments encoded by the HIV-1 Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes.In some embodiments, the plurality of polypeptide segments consists of only polypeptide segments encoded by the HIV-1 Pol gene and, for example, does not include polypeptide segments encoded by the HIV-1 Env, Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes. In various embodiments, the plurality of polypeptide segments does not contain segments encoded by one, two, three, or four of the HIV Tat, Rev, Vif, Vpr, and / or Vpu genes.

[0355] With respect to the number of polypeptide segments assembled, linked, or concatenated into a single fusion polypeptide, in various embodiments, the fusion polypeptide consists of at least 5 and up to 40 polypeptide segments, for example, 5 polypeptide segments and up to 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, or 40 polypeptide segments. Optionally, the polypeptide segments can be arranged in the same order or in an order different from that of the naturally occurring protein.

[0356] Regarding regions of polypeptides encoded by HIV-1 genes selected as polypeptide segments comprising a fusion polypeptide, in various embodiments, the polypeptide segment is derived from a conserved region of a population of viral proteome sequences. In some embodiments, the conserved region is greater than 80%, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% conserved among HIV-1 species, as determined, for example, among patients and / or within a 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 the same amino acid segment or subsequence (e.g., a segment 9 amino acids in length or a 9mer segment) as the most prevalent at a given amino acid segment or subsequence position, where the amino acid segment or subsequence position is determined with respect to a reference sequence (e.g., the HIV-1 HXB2 polypeptide sequence (e.g., SEQ ID NOs: 403 - 406)). In various embodiments, the conserved region is conserved among one or more of the HIV-1 clades within group M, e.g., one or more of HIV-1 clades A - K, e.g., one or more of clades A, B, C, D, and G, e.g., among HIV-1 group M, clade B, and recombinant forms thereof, e.g., CRF01_AE. In some embodiments, the plurality of polypeptide segments comprises at least 2 polypeptide segments selected from SEQ ID NOs: 1 - 344, 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 segments, e.g., the polypeptide segments identified in Table 2.In some embodiments, the plurality of polypeptide segments comprises at least two polypeptide segments selected from SEQ ID NOs: 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, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more polypeptide segments, for example, the polypeptide segments specified in Table 3.The start and end positions are specified in Table 1 with respect to the HIV-1 HXB2 reference polypeptide provided as SEQ ID NOs: 403 to 406 in this specification, GenBank Accession No. K03455 (ncbi.nlm.nih.gov / nuccore / K03455). [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

Table 3-5

Table 3-6

[0357] Regarding the length ranges of the individual polypeptides or peptide segments, in various embodiments, each polypeptide segment is at least 8 amino acids in length and up to about 250 amino acids in length. For example, it can be from at least 8 amino acids in length up to a maximum of 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 segment is at least 8 amino acids in length and up to about 35 amino acids in length. For example, it can be from at least 8 amino acids in length up to a maximum of 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 segment is at least 15 amino acids in length and up to about 30 amino acids in length. For example, it can be from at least 15 amino acids in length up to a maximum of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0358] Regarding the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full length of the fusion polypeptide is at least about 350 amino acids and up to about 1000 amino acids, for example, at least about 350 amino acids and up to 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, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 amino acids. Regarding the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full length of the fusion polypeptide is at least about 350 amino acids and up to about 800 amino acids, for example, at least about 350 amino acids and up to 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 full length of the fusion polypeptide is 800 amino acids or less, for example, 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, 705, or 700 amino acids.

[0359] Generally, the fusion polypeptide is immunogenic in that it can induce an immune response against, for example, HIV-1 in humans. In some embodiments, the fusion polypeptide can optionally, for example, in combination with one or more additional therapeutic agents as described herein, induce a protective or therapeutically effective immune response against HIV-1 in humans, for example, prevent HIV-1 infection in uninfected individuals, or in a therapeutic setting, induce immune-mediated control of HIV-1 or induce an immune response sufficient to eradicate HIV-1 in infected individuals. The immunogenicity of the fusion polypeptide can be evaluated and demonstrated in in vitro and in vivo assays as described herein. For example, the immunogenicity of the fusion polypeptide can be demonstrated by in vitro assays including CD4+ and / or CD8+ T cell activation (including cytokine expression and target killing assays) or proliferation assays. T cells can be activated by exposure to antigen presenting cells (APCs) (e.g., dendritic cells, e.g., monocyte-derived dendritic cells) transfected with a polynucleotide encoding the fusion polypeptide. Such assays are known in the art and are described herein. The immunogenicity of the fusion polypeptide can also be demonstrated in in vivo animal models, for example, by administering to mice or non-human primates transgenic for one or more human HLA molecules (available from Jackson Laboratories or Taconic) and evaluating CD4+ and / or CD8+ T cell activation (including serum cytokine levels) or proliferation. In various embodiments, one, two, three, or more of each polypeptide segment includes or consists of one or more predicted T cell epitopes determined, for example, computationally or experimentally. In some embodiments, the fusion polypeptide includes one or more polypeptide segments 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) within a single subject or among multiple subjects.In some embodiments, the fusion polypeptide comprises at least human A. * 0201 It comprises one or more polypeptide segments that bind to or are presented by HLA class I molecules. In some embodiments, the fusion polypeptide binds to or is presented by, for example, one or more human HLA class I and / or class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles) within a single subject, and 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 8mer, 9mer, and / or 10mer polypeptide segments. In some embodiments, the fusion polypeptide is 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), for example, within a single subject, and 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 segments each 15 - 30 amino acids in length. Linker polypeptide segment

[0360] Optionally, one or more of the polypeptide segments can be in direct contact with, or fused to, an adjacent segment, or can be joined, connected, or linked to an adjacent segment by one or more peptide linkers. In various embodiments, one or more peptide linkers are selected from, for example, one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, a Nef-linking sequence, and combinations thereof, within the linker or within the full-length fusion polypeptide. Exemplary fusion protein linkers that can be used in the fusion polypeptides of the invention to connect one or more polypeptide segments are described, for example, in Chen, et al., Adv Drug Deliv Rev. (2013) 65(10):1357-1369. In some embodiments, a 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, such as GGS, GSG or GGGS (SEQ ID NO: 421) is used.

[0361] In some embodiments, the cleavable linker is selected from 2A cleavable peptides. Exemplary 2A cleavable peptides that can be used in the fusion polypeptides of the present invention to connect one or more polypeptide segments 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 segments include, but are not limited to, 2A cleavage sequences (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna 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 sequences (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)) are combined or fused with 2A cleavable peptides (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A)) within a single linker. See, for example, Chng, et al., mAbs (2015) 7:2, 403-412.In various embodiments, the 2A cleavable linker comprises, consists of, or consists essentially 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, or consists of, the amino acid sequence of 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). Optionally, in certain embodiments, the furin recognition / cleavage sequence can be positioned at either the N-terminus or the 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 cleavable linker that naturally occurs in the 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, and the Nef-linker sequence comprises, consists of, or consists essentially 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 link or connect one or more polypeptide segments in a fusion polypeptide are provided in Table 4. [Table 4] A polypeptide segment encoded by the HIV-1 Gag gene

[0362] In various embodiments, the fusion polypeptide comprises one or more segments of one or more viral proteins encoded by the HIV-1 Gag gene, or fragments or subsequences 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 include any p6 component.In some embodiments, the plurality of polypeptide segments comprise at least two polypeptide segments having an amino acid sequence selected from SEQ ID NOs: 68-146 and 339-342; SEQ ID NOs: 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 NOs: 76, 77, 86, 87 and 92-124; SEQ ID NOs: 76, 77, 86, 87, 94 and 95; SEQ ID NOs: 76, 86 and 94; SEQ ID NOs: 77, 87 and 95; SEQ ID NOs: 68-79 and 92-124; SEQ ID NOs: 70-71, 76-77 and 94-95; SEQ ID NOs: 78, 79, 96, 99, 100, 107, 108, 113, 114, 121, 122, 123, 124, 137 and 138; SEQ ID NOs: 78, 99, 107, 113, 121, 123 and 137; SEQ ID NOs: 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; SEQ ID NOs: 78, 90, 97, 105, 111, 117, 119 and 137; and SEQ ID NOs: 78 and 137, or consisting of at least 2, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more segments.

[0363] In some embodiments, the fusion polypeptide comprises, or consists of, at least two polypeptide segments corresponding to HIV-1 Gag amino acid sequences at amino acid residue positions selected from 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, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more segments, and the amino acid positions are with respect to SEQ ID NO: 404. In certain embodiments, the fusion polypeptide does not comprise one, two, three, four, five, or more polypeptide segments corresponding to, or consisting of, an HIV-1 Gag amino acid sequence or a subsequence thereof at amino acid residue positions selected from 1-30, 54-127, 138-146, 370-428, and 445-500, and the amino acid positions are with respect to SEQ ID NO: 404. In some embodiments, the plurality of polypeptide segments do not comprise one, two, three, four, five, or more polypeptide segments comprising, or consisting of, a sequence or a subsequence thereof 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 the HIV-1 Gag amino acid sequences of SEQ ID NOs: 444-448, or the fusion proteins described herein do not comprise them.As used herein, the numbering of a given amino acid polymer or nucleic acid polymer "corresponds to," "corresponding to," or "relative to" the selected or referenced amino acid polymer or nucleic acid polymer when the position of any given polymer component (e.g., an amino acid, nucleotide, commonly also referred to as a "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 the actual numerical position of the component in the given polymer. A polypeptide segment encoded by the HIV-1 Nef gene

[0364] In some embodiments, the fusion polypeptide comprises one or more segments of a viral protein encoded by the HIV-1 Nef gene. In some embodiments, the plurality of polypeptide segments are SEQ ID NOs: 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; SEQ ID NOs: 153, 154, 155, 156, 157, 158, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172 and 173; SEQ ID NO: 153 and 165, and at least one polypeptide segment comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 153, for example, 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 segments.

[0365] In some embodiments, the fusion polypeptide comprises, or consists of, at least two polypeptide segments corresponding to HIV-1 Nef amino acid sequences at amino acid residue positions selected from 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, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more segments, and the amino acid positions are with respect to SEQ ID NO: 405. In certain embodiments, the fusion polypeptide does not comprise one, two, three, or more polypeptide segments corresponding to the HIV-1 Nef amino acid sequence or a subsequence thereof at amino acid residue positions selected from 1-63, 103-116, and 155-206, and the amino acid positions are with respect to SEQ ID NO: 405. In some embodiments, the plurality of polypeptide segments do not comprise one, two, three, or more polypeptide segments comprising, or consisting of, a sequence or a subsequence thereof 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 the HIV-1 Nef amino acid sequences of SEQ ID NOs: 449-451, or the fusion proteins described herein do not comprise them. Fusion polypeptide having polypeptide segments encoded by the HIV-1 Gag and Nef genes

[0366] In some embodiments, the fusion polypeptide comprises or consists of one or more segments of viral proteins encoded by the HIV-1 Gag and Nef genes, and for example, does not contain one or more polypeptide segments encoded by the HIV-1 Env, Pol, Tat, Rev, Vif, Vpr or Vpu genes. In some embodiments, the fusion polypeptide comprises or consists of at least two polypeptide segments selected from the amino acid sequences of SEQ ID 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, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more segments.

[0367] In some embodiments, the fusion polypeptide comprises or consists of the following polypeptide segments, in N-terminal to C-terminal order, optionally joined or connected by one or more linkers of SEQ ID NOs: 70, 76, 94, 151 and 161; or SEQ ID NOs: 71, 77, 95, 152 and 162.

[0368] 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.

[0369] Modifications can be made to the structure of the fusion polypeptides described herein and the polynucleotides encoding such fusion polypeptides, and functional molecules encoding variant or derivative polypeptides that still have the desired (e.g., immunogenic) properties may be obtained. When it is desirable to alter the amino acid sequence of a polypeptide to create an equivalent of the fusion polypeptides described herein, or a further improved variant or portion, one of ordinary skill in the art will typically alter one or more of the codons of the coding DNA sequence.

[0370] For example, certain amino acids can be substituted for other amino acids in a protein structure without apparently losing the ability to bind to other polypeptides (e.g., antigens) or cells. Since this is the binding ability and nature of the protein that defines the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in the underlying DNA coding sequence, and a protein with similar properties can still be obtained. Accordingly, it is contemplated that various changes can be made to the polypeptide sequence of the disclosed fusion polypeptides, or to the corresponding DNA sequences encoding such fusion polypeptides without an apparent loss of their biological utility or activity.

[0371] Often, polypeptide variants contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, such that one of ordinary skill in peptide chemistry would expect the secondary structure and hydropathicity properties of the polypeptide to not substantially change.

[0372] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the nucleotide or amino acid sequences in the two sequences are the same when the two sequences are aligned to maximize correspondence as described below. Comparisons between two sequences are typically performed by comparing 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, usually 30 to about 75, 40 to about 50, where the sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0373] Optimal alignment of the arrays for comparison may be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc. (Madison, WI)) using default parameters. This program implements several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (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, D.G. and Sharp, P.M. (1989) CABIOS 5:151 - 153; Myers, E.W. and Muller W. (1988) CABIOS 4:11 - 17; Robinson, E.D. (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406 - 425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA 80:726 - 730.

[0374] Alternatively, the optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the method of searching for similarity of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by 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 by inspection.

[0375] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described below: Altschul et al. (1977) “Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine the percent sequence identity of the polynucleotides and polypeptides described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0376] In an exemplary case, for a nucleotide sequence, the cumulative score can be calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). The extension of a word hit in each direction stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score residues; 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 word length (W) of 11 and an expectation value (E) of 10 as defaults, and BLOSUM62 is used as the scoring matrix (see the following: Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) for alignment, (B) 50, expectation value (E) 10, M = 5, N = -4, and comparison of both strands as defaults.

[0377] For an amino acid sequence, the cumulative score can be calculated using a scoring matrix. The extension of a word hit in each direction stops when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0378] In one approach, the "percentage of sequence identity" is determined by comparing two sequences optimally aligned over a comparison window of at least 20 positions. The portion of the polynucleotide or polypeptide sequence in the comparison window may include from 20 percent or less, usually from 5 to 15 percent, or from 10 to 12 percent additions or deletions (i.e., gaps) as compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0379] As used herein, the term "polypeptide variant" is typically a different polypeptide from the polypeptides specifically disclosed herein, with one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above-described polypeptide sequences herein, by evaluating one or more biological activities of the polypeptides described herein, and / or by using any of several techniques well known in the art. The term "variant" may also refer to any naturally occurring or genetically engineered molecule that contains one or more nucleotide or amino acid mutations. A polypeptide segment encoded by the HIV-1 Env gene

[0380] In some embodiments, the fusion polypeptide comprises one or more segments 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.

[0381] In various embodiments, the fusion polypeptide comprises at least two polypeptide segments, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more segments, comprising or consisting of amino acid sequences selected from SEQ ID NOs: 1-67 and 338; SEQ ID NOs: 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 NOs: 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 NOs: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 28, 29, 30, 37, 38, 41 and 42; SEQ ID NOs: 28, 29, 30 and 41-56; SEQ ID NOs: 28, 29, 41 and 42; SEQ ID NOs: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 37 and 38; SEQ ID NOs: 4, 5, 11, 12, 37 and 38; SEQ ID NOs: 6, 7, 15, 16, 21, 22, 30, 60 and 61; SEQ ID NOs: 6, 15, 21, 30 and 60; SEQ ID NOs: 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 NOs: 1, 10, 19, 27, 55, 56 and 57, and SEQ ID NOs: 6, 15 and 60.

[0382] In various embodiments, the fusion polypeptide comprises, or consists of, at least two polypeptide segments corresponding to HIV-1 Env amino acid sequences at amino acid residue positions selected from 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, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more segments, and the amino acid positions are with respect to SEQ ID NO: 403. In certain embodiments, the fusion polypeptide does not comprise, or consist of, 1, 2, 3, 4, 5, 6, or more polypeptide segments corresponding to HIV-1 Env amino acid sequences at amino acid residue positions selected from 1-27, 53-58, 84-112, 138-234, 269-474, 490-501, 611-856, or a subsequence thereof, and the amino acid positions are with respect to SEQ ID NO: 403. In some embodiments, the plurality of polypeptide segments do not comprise, or consist of, 1, 2, 3, 4, 5, or more polypeptide segments comprising, or consisting of, a sequence or a subsequence thereof 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 the HIV-1 Env amino acid sequences of SEQ ID NOs: 437-443, or the fusion proteins described herein do not comprise them. Polypeptide segment encoded by the HIV-1 Pol gene

[0383] In some embodiments, the fusion polypeptide comprises one or more segments of one or more viral proteins encoded by the HIV-1 Pol gene. In various embodiments, 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).

[0384] In some embodiments, the plurality of polypeptide segments are set forth in SEQ ID NOs: 174-337 and 343-344; SEQ ID NOs: 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: 180, 181, 186, 187, 221, 222, 294, 295, 307, 308, 321, and 322;Array numbers 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; Array numbers 176, 177, 188, 189, 213, 214, 223, 224, 259, 260, 282, 283, 294, 295, 305, 306, 319, and 320; Array numbers 180, 181, 186, 187, 221, 222, 294, 295, 321 and 322; Array numbers 182 to 202, 292 to 302, 305, 306; Array numbers 188, 189, 294, 295, 305, 306; Array numbers 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, 242, 243, 244, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 282, 283, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337; Array numbers 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319, and 320;Array numbers 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; Array numbers 192, 201, 215, 217, 219, 229, 230, 240, 241, 243, 265, 276, 298, 302, 311, 327, 331, 333, and 336; Array numbers 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, 343, 344, 375, and 376; Array numbers 190, 197, 209, 210, 211, 225, 227, 234, 238, 261, 296, 300, 303, 323, 325, 329, 334, and Array numbers 192, 215, 217, 219, 229, 230, 276, 298, 302, 327, 331, 333, and 336 selected amino acid sequences, or at least two polypeptide segments consisting of them, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more segments. ;

[0385] In some embodiments, the fusion polypeptide comprises, or consists of, at least two polypeptide segments corresponding to HIV-1 Pol amino acid sequences at amino acid residue positions selected from 144 - 168, 152 - 160, 291 - 315, 326 - 350, 328 - 352, 330 - 354, 333 - 354, 334 - 342, 336 - 344, 338 - 346, 374 - 398, 380 - 404, 382 - 390, 388 - 396, 399 - 423, 400 - 424, 406 - 430, 553 - 577, 642 - 666, 650 - 658, 759 - 783, 767 - 775, 768 - 792, 776 - 784, 834 - 858, 940 - 964, 947 - 971, 948 - 956, 948 - 972, 955 - 963, 956 - 964, 980 - 1003, and 988 - 996, 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 segments, and the amino acid positions are with respect 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 segments corresponding to HIV-1 Pol amino acid sequences at amino acid residue positions selected from 11 - 55, 118 - 128, 321 - 325, 355 - 366, 432 - 541, 607 - 641, 667 - 682, 709 - 746, 828 - 833, 921 - 930, or a subsequence thereof, and the amino acid positions are with respect to SEQ ID NO: 406. In some embodiments, the plurality of polypeptide segments do not comprise 1, 2, 3, 4, 5, or more polypeptide segments comprising, or consisting of, a sequence or a subsequence thereof 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 the HIV-1 Pol amino acid sequences of SEQ ID NOs: 452 - 461, or the fusion proteins described herein do not comprise them.

[0386] In some embodiments, the fusion polypeptide comprising a polypeptide segment encoded by the HIV-1 Pol gene does not contain 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 contain 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 contain one or more amino acid sequences selected from SEQ ID NOs: 209, 210, 211, 212, 213, 214, 343, and 344. A fusion polypeptide having polypeptide segments encoded by the HIV-1 Env and Pol genes

[0387] In some embodiments, the fusion polypeptide comprises or consists of one or more segments of viral proteins encoded by the HIV-1 Env and Pol genes, and for example, does not contain one or more polypeptide segments encoded by the HIV-1 Gag, Nef, Tat, Rev, Vif, Vpr, or Vpu genes.

[0388] In some embodiments, the fusion polypeptide is set forth in SEQ ID NOs: 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, 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;At least two polypeptide segments comprising, or consisting of, an amino acid sequence selected from SEQ ID NOs: 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 37, 38, 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, 242, 243, 244, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 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, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more segments. ;

[0389] In some embodiments, the fusion polypeptide comprises polypeptide segments of SEQ ID NO: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213, and 37; SEQ ID NO: 188, 305, 28, 41, and 294; SEQ ID NO: 4, 176, 11, 319, 259, 282, 223, 213, and 37; SEQ ID NO: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214, and 38; SEQ ID NO: 189, 306, 29, 42, and 295; 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; SEQ ID 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, optionally linked or connected by one or more linkers in order from the N-terminus to the C-terminus.

[0390] In some embodiments, the fusion polypeptide comprises, or consists of, an amino acid sequence of any one of SEQ ID NOs: 357 - 366 and 407 - 410, 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: 357 - 366 and 407 - 410, wherein the percent identity is over the full length of SEQ ID NOs: 357 - 366 and 407 - 410. A fusion polypeptide having polypeptide segments encoded by the HIV-1 Gag, Nef, and Pol genes

[0391] In some embodiments, the fusion polypeptide comprises or consists of one or more segments of viral proteins encoded by the HIV-1 Gag, Nef, and Pol genes, and, for example, does not include one or more polypeptide segments encoded by the HIV-1 Env, Tat, Rev, Vif, Vpr, or Vpu genes.

[0392] In some embodiments, the fusion polypeptide comprises or consists of at least two polypeptide segments having amino acid sequences selected from SEQ ID NOs: 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, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 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, 337, 339, 340, 341, and 342, and SEQ ID NOs: 76, 77, 86, 87, 94, 95, 151, 152, 181, 182, 186, 187, 221, 222, 294, 195, 307, 308, 321, 322, and includes, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more segments.

[0393] In some embodiments, the fusion polypeptide comprises, in order from the N-terminus to the C-terminus, optionally linked or connected by one or more linkers, the polypeptides of SEQ ID NOs: 76, 86, 94, 180, 186, 221, 294, 307, 321, and 151; or the polypeptide segments of SEQ ID NOs: 77, 87, 95, 181, 187, 222, 295, 308, 322, and 152.

[0394] In some embodiments, the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 345 - 350 and 422 - 424, 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: 345 - 350 and 422 - 424, and the percent identity is over the full length of SEQ ID NOs: 345 - 350 and 422 - 424.

[0395] In some embodiments, the fusion polypeptide comprises or consists of an amino acid sequence of 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, and the percent identity is over the full length of SEQ ID NOs: 527 - 528. A fusion polypeptide having a polypeptide segment encoded by HIV-1 Env, Gag, Nef, and Pol and predicted to bind to or be presented by the human HLA A * 0201 molecule

[0396] As described herein, the inventors have shown that human HLA A *A fusion polypeptide having a plurality of computationally predicted polypeptide segments that bind to or are presented by the 0201 molecule was designed. Generally, the selected or included polypeptide segments in such fusion polypeptides include 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 have an IC50 value of less than about 1,000 nM for human HLA A * is predicted to bind to the 0201 molecule. In certain embodiments, the selected or included polypeptide segments in such fusion polypeptides include 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 within the top 5% percentile rank in the population of polypeptide segments predicted to bind to human HLA A * is predicted to bind to the 0201 molecule. In some embodiments, the fusion polypeptide comprises or consists of segments of viral proteins encoded by the Gag, Pol, Env, and Nef genes, and each of the plurality of polypeptide segments can bind to or be presented by human HLA allele A * 0201.

[0397] In some embodiments, each of the plurality of polypeptide segments is 8 to 35 amino acids in length, e.g., 9 to 34 amino acids in length, e.g., 9 to 25 amino acids in length.

[0398] In some embodiments, the fusion polypeptide is set forth in 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; SEQ ID NOs: 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 NOs: 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, 114, 117, 118, 119, 120, 121, 122, 123, 124, 137, 138, 153, 154, 155, 156, 157, 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;At least two polypeptide segments comprising or consisting of an amino acid sequence selected from SEQ 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, 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, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, or more segments. ;

[0399] In some embodiments, the fusion polypeptide comprises polypeptide segments of 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; SEQ ID 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, connected or joined in order from the N-terminus to the C-terminus, optionally by one or more linkers.

[0400] 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, and the percent identity is over the full length of SEQ ID NOs: 367-377 and 411. Signal or leader sequence

[0401] In various embodiments, the fusion polypeptide includes a signal sequence or signal peptide, for example, to direct intracellular transport of the fusion polypeptide to the proteasome or lysosomal compartment. In various embodiments, the fusion polypeptide includes a signal sequence at the N-terminus and / or 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 derived from a source protein selected from serum proteins, cytokines, chemokines, chaperone proteins, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In some embodiments, the signal peptide or leader sequence is colony stimulating factor 2 (CSF2, GM-CSF), tissue type plasminogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C 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 an N-terminal and a C-terminal signal sequence from LAMP-1, for example, SEQ ID NO: 399 and SEQ ID NO: 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 that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 393-402 and 412-413, and the percent identity is over the full length of SEQ ID NOs: 393-402 and 412-413. Polynucleotide encoding the fusion polypeptide

[0402] Provided are polynucleotides encoding the 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, e.g., 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).

[0403] Polynucleotides comprising nucleotide sequences encoding any of the fusion polypeptides provided herein, as well as expression cassettes, expression vectors, and viral vectors comprising such nucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells, are provided herein. In various embodiments, the polynucleotide is DNA, cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotide comprises an alphavirus self-replicating or self-amplifying replicon RNA (RepRNA). Self-replicating RNA and self-amplifying replicon RNA as modes of vaccine delivery are described, e.g., in Tews, et al., Methods Mol Biol. (2017) 1499:15-35; Demoulins, 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.

[0404] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides, including RNA, cDNA, genomic DNA, and both sense and antisense strands of the foregoing synthetic forms and mixed polymers. As used herein, the term nucleic acid molecule may be interchangeable with the term polynucleotide. In some embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide, or modified form of either 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, e.g., cDNA or mRNA, may contain either or both natural and modified nucleotides joined together by natural and / or non-natural nucleotide linkages. A nucleic acid molecule may be chemically or biochemically modified, or contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. References to nucleic acid sequences include the complement thereof unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to include the complementary strand having the complementary sequence.This term also includes codon-optimized polynucleotides for improved expression in a desired viral expression vector or host cell.

[0405] As used herein, "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

[0406] "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes nucleic acid molecules contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. "Isolated nucleic acid encoding a polypeptide segment or a fusion polypeptide" refers to such a polypeptide segment or fusion polypeptide, including such nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules encoding such nucleic acid molecules present at one or more locations within a host cell.

[0407] "Polynucleotide variant", as the term is used herein, is typically a different polynucleotide from the polynucleotides specifically disclosed herein, with one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be generated synthetically, for example, by modifying one or more of the polynucleotide sequences described herein, by assessing one or more biological activities of the encoded polypeptides described herein, and / or using any of several techniques well known in the art.

[0408] In some embodiments, the nucleic acid molecule is codon-biased to enhance expression in a desired host cell, such as a human cell, mammalian cell, yeast cell, plant cell, insect cell, or bacterial cell, such as an E. coli cell. Accordingly, a polynucleotide encoding the fusion polypeptide described herein is provided, wherein the polynucleotide is codon-optimized, contains a substituted heterologous signal sequence, and / or has removed mRNA instability elements. Methods for generating codon-biased nucleic acids can be performed, for example, by applying the methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132, and 6,794,498. Preferred codon usage frequencies for the expression of a fusion polypeptide comprising an HIV-1 polypeptide segment from a desired viral expression vector and / or in a desired host cell are provided, for example, at kazusa.or.jp / codon / , and genscript.com / tools / codon-frequency-table. And genscript.com / tools / codon-frequency-table.

[0409] In some embodiments, a polynucleotide encoding a fusion polypeptide as described herein has 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%, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 414-418, wherein the percent identity is over the full length of SEQ ID NOs: 414-418.

[0410] In some embodiments, a polynucleotide encoding a fusion polypeptide as described herein has 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% identity, or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 524 - 526, and the percent identity is over the full length of SEQ ID NOs: 524 - 526.

[0411] In various embodiments, one or more polynucleotides 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, or to any one of SEQ ID NOs: 345 - 377, 407 - 411, 422 - 424, and 430 - 435.

[0412] In various embodiments, the viral vector or viral expression vector 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 sequences of SEQ ID NO: 345 and 346; SEQ ID NO: 347 and 348; SEQ ID NO: 349 and 350; SEQ ID NO: 351 and 352; SEQ ID NO: 430 and 352; SEQ ID NO: 357 and 358; SEQ ID NO: 360 and 362; SEQ ID NO: 359 and 361; SEQ ID NO: 351 and 357; SEQ ID NO: 351 and 358; SEQ ID NO: 351 and 359; SEQ ID NO: 351 and 360; SEQ ID NO: 351 and 361; SEQ ID NO: 351 and 362; SEQ ID NO: 351 and 407; SEQ ID NO: 351 and 408; SEQ ID NO: 351 and 409; SEQ ID NO: 351 and 410; SEQ ID NO: 352 and 357; SEQ ID NO: 352 and 358; SEQ ID NO: 352 and 359; SEQ ID NO: 352 and 360; SEQ ID NO: 352 and 361; SEQ ID NO: 352 and 362; SEQ ID NO: 352 and 407; SEQ ID NO: 352 and 408; SEQ ID NO: 352 and 409; SEQ ID NO: 352 and 410; SEQ ID NO: 430 and 357; SEQ ID NO: 430 and 358; SEQ ID 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, and encodes two or more fusion proteins.

[0413] 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 contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mismatch, or fewer amino acid mismatches, relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains 15 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains 10 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains 5 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527.In some embodiments, the polynucleotide encodes a fusion protein that contains four or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains three or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains two or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527. In some embodiments, the polynucleotide encodes a fusion protein that contains one or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527.

[0414] 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 contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mismatch, or fewer, relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 15 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 10 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 5 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528.In some embodiments, the polynucleotide encodes a fusion protein that contains 4 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 3 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 2 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528. In some embodiments, the polynucleotide encodes a fusion protein that contains 1 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528.

[0415] In various embodiments, 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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, and 430-435.

[0416] 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%, 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. In some embodiments, the polynucleotide is at least 100% identical to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0417] 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%, 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, a polynucleotide that contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer, relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0418] 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, a polynucleotide comprising 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer mismatches, compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 15 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 10 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 5 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 4 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 3 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising 2 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 526.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0419] 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%, 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, a polynucleotide that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0420] 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%, 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, a polynucleotide that contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer, relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0421] 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, a polynucleotide comprising 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, a polynucleotide comprising 15 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising 10 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising 5 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising 4 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising 3 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising 2 or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0422] 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, a polynucleotide that contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer than that, with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 15 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 10 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 5 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 4 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 3 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide that contains 2 or fewer nucleotide mismatches with respect to the nucleic acid sequence of SEQ ID NO: 521.In some embodiments, a polynucleotide comprising one or fewer nucleotide mismatches to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, a polynucleotide comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 521.

[0423] Optionally, in certain embodiments, the 3’ end of the polynucleotide encoding the fusion polypeptide described herein comprises one or more tandem stop codons, e.g., two or more tandem TAG (“amber”), TAA (“ochre”), or TGA (“opal” or “amber”) stop codons. The plurality of tandem stop codons may be the same or different.

[0424] As described herein, there is further provided an expression cassette comprising a polynucleotide encoding a fusion polypeptide 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 cytomegalovirus major immediate-early (CMV), chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), murine cytomegalovirus (murine CMV), Chinese hamster elongation factor-1α (CHEF-1α), and CMV enhancer fused to phosphoglycerate kinase (PGK). Expression cassettes, vectors, viral vectors, and host cells

[0425] There is further provided a vector comprising one or more fusion polypeptides described herein, or one or more polynucleotides encoding an expression cassette comprising such a polynucleotide. The vector can be a recombinant vector of any type, such as an expression vector. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), and vectors derived from bacteriophages, or plant or animal (including human) viruses. The vector can include an origin of replication recognized by the proposed host cell, and in the case of an expression vector, can include a promoter and other regulatory regions recognized by the host cell. In additional embodiments, the vector comprises one or more polynucleotides encoding one or more fusion polypeptides of the present disclosure operably linked to a promoter and optionally additional regulatory elements. Certain vectors can replicate autonomously in the host into which they are introduced (e.g., a vector having a bacterial origin of replication can replicate in bacteria). Other vectors can integrate into the genome of the host upon introduction into the host and are thereby replicated with the host genome. Vectors include, but are not limited to, those suitable for the recombinant production of the fusion polypeptides disclosed herein.

[0426] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of the introduced host cell. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors can induce the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as expression vectors.

[0427] The term "operably linked" generally refers to two or more nucleic acid sequence elements that are physically linked and functionally related to each other. As an example, a promoter is operably linked to a coding sequence if the promoter can initiate or regulate the transcription or expression of the coding sequence, in which case the coding sequence is to be understood as being "under the control of the promoter".

[0428] The choice of vector depends on the recombinant procedures to be followed and the host to be used. Introduction of the vector into the host cell can be effected, inter alia, by calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofectamine transfection, electroporation, viral infection, or administration to a subject, as described herein. The vectors can replicate autonomously or can replicate together with the chromosome into which they are integrated. In certain embodiments, the vector contains one or more selectable markers. The choice of marker may depend on the host cell to be selected. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, the thymidine kinase gene from herpes simplex virus (HSV-TK), and the mouse-derived dihydrofolate reductase gene (dhfr). Also encompassed by the present disclosure are vectors containing one or more nucleic acid molecules encoding the fusion polypeptides described herein, operably linked to one or more nucleic acid molecules encoding a protein or peptide that can be used to isolate the fusion polypeptide ("purification tag"). These proteins or peptides include, but are not limited to, the FLAG tag (DYKDDDDKL; SEQ ID NO: 436), glutathione-S-transferase, maltose-binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and β-galactosidase.

[0429] In other embodiments, the vector used is pcDNA™ 3.1+ (ThermoFisher, MA).

[0430] 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, while a viral vector refers to a viral particle. Optionally, the viral expression vector or the viral vector can be a DNA virus or an RNA virus, including a self-replicating RNA virus. Self-replicating RNA viruses include alphaviruses, for example, as described 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 the viral vector is derived 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 derived from a viral family selected from the group consisting of Adenoviridae (e.g., adenovirus, adeno-associated virus), Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus, Calomys callosus arenavirus (also known as Pichinde mammarenavirus)), Herpesviridae (e.g., cytomegalovirus, herpes virus, e.g., HSV-1), Parvoviridae (e.g., parvovirus H1), Poxviridae (e.g., vaccinia virus, e.g., modified vaccinia Ankara (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., vesiculovirus including Maraba virus and Vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, e.g., self-replicating alphavirus; Sindbis virus), Enteroviridae (e.g., echovirus). Exemplary modified vaccinia virus vectors and viral vectors for use in expressing the fusion polypeptides of the present invention are described, for example, in International Publication No. WO 2019 / 134049.

[0431] In some embodiments, the viral expression vector or viral vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) (NCBI:txid11623), Calomys callosus arenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus) (NCBI:txid2169993), Guanarito virus (GTOV) (NCBI:txid45219), Argentine mammarenavirus (also known as Junin virus (JUNV)) (NCBI:txid2169991), Lassa virus (LASV) (NCBI:txid11620), Lujo virus (LUJV) (NCBI:txid649188), Machupo virus (MACV) (NCBI:txid11628), Brazilian mammarenavirus (also known as Sabiá 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 lymphocytic choriomeningitis mammarenavirus (LCMV), or Calomys callosus arenavirus (also known as Pichinde mammarenavirus 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 International Publication Nos. WO 2009 / 083210, WO 2015 / 183895, WO 2016 / 075250, WO 2017 / 198726, and U.S. Patent No. 9,943,585.

[0432] In some embodiments, the viral expression vector or viral vector is an adenoviral vector, e.g., derived from a human adenovirus or a simian adenovirus (e.g., chimpanzee adenovirus, gorilla adenovirus, or rhesus adenovirus). In various embodiments, the adenoviral vector is selected from adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (e.g., 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), 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), ChAd147 (AdC147)), gorilla adenovirus (e.g., GC44, GC45, GC46) and rhesus adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66).Exemplary chimpanzee, gorilla, and rhesus adenovirus vectors that can be used as delivery and expression vehicles for the fusion polypeptides described herein are described, for example, in International Publication Nos. 2019 / 076880, 2019 / 076877, Andrabie 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.

[0433] In various embodiments, the viral expression vector or viral vector is non-replicable (i.e., replication-deficient or replication-impaired), or has a reduced or decreased ability to replicate (i.e., is attenuated for replication) compared to a wild-type viral vector, or is replicable.

[0434] In various 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 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, or to any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435.

[0435] 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% or 100% identical to the amino acid sequences of SEQ ID NO: 345 and 346; SEQ ID NO: 347 and 348; SEQ ID NO: 349 and 350; SEQ ID NO: 351 and 352; SEQ ID NO: 430 and 352; SEQ ID NO: 357 and 358; SEQ ID NO: 360 and 362; SEQ ID NO: 359 and 361; SEQ ID NO: 351 and 357; SEQ ID NO: 351 and 358; SEQ ID NO: 351 and 359; SEQ ID NO: 351 and 360; SEQ ID NO: 351 and 361; SEQ ID NO: 351 and 362; SEQ ID NO: 351 and 407; SEQ ID NO: 351 and 408; SEQ ID NO: 351 and 409; SEQ ID NO: 351 and 410; SEQ ID NO: 352 and 357; SEQ ID NO: 352 and 358; SEQ ID NO: 352 and 359; SEQ ID NO: 352 and 360; SEQ ID NO: 352 and 361; SEQ ID NO: 352 and 362; SEQ ID NO: 352 and 407; SEQ ID NO: 352 and 408; SEQ ID NO: 352 and 409; SEQ ID NO: 352 and 410; SEQ ID NO: 430 and 357; SEQ ID NO: 430 and 358; SEQ ID 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.

[0436] 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% 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 that comprises 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 relative 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 comprises 15 or fewer amino acid mismatches relative 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 comprises 10 or fewer amino acid mismatches relative 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 comprises 5 or fewer amino acid mismatches relative 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 comprises 4 or fewer amino acid mismatches relative 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 comprises 3 or fewer amino acid mismatches relative 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 comprises 2 or fewer amino acid mismatches relative 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 comprises 1 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527.

[0437] 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 comprising 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 relative 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 comprising 15 or fewer amino acid mismatches relative 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 comprising 10 or fewer amino acid mismatches relative 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 comprising 5 or fewer amino acid mismatches relative 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 comprising 4 or fewer amino acid mismatches relative 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 comprising 3 or fewer amino acid mismatches relative 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 comprising 2 or fewer amino acid mismatches relative 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 comprising 1 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528.

[0438] 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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, and 430-435.

[0439] 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 that comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer, relative 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 contains at least 15 or fewer nucleotide mismatches relative 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 contains at least 10 or fewer nucleotide mismatches relative 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 contains at least 5 or fewer nucleotide mismatches relative 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 contains at least 4 or fewer nucleotide mismatches relative 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 contains at least 3 or fewer nucleotide mismatches relative 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 contains at least 2 or fewer nucleotide mismatches relative 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 contains at least 1 or fewer nucleotide mismatches relative 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 contains the codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0440] 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 that contains 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 expression cassette, expression vector, viral expression vector or viral vector comprises a polynucleotide comprising at most 15 nucleotide mismatches relative 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 comprising at most 10 nucleotide mismatches relative 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 comprising at most 5 nucleotide mismatches relative 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 comprising at most 4 nucleotide mismatches relative 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 comprising at most 3 nucleotide mismatches relative 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 comprising at most 2 nucleotide mismatches relative 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 comprising at most 1 nucleotide mismatch relative 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 comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0441] 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 that contains 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 expression cassette, expression vector, viral expression vector or viral vector comprises a polynucleotide comprising at least 15 or fewer nucleotide mismatches relative 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 comprising at least 10 or fewer nucleotide mismatches relative 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 comprising at least 5 or fewer nucleotide mismatches relative 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 comprising at least 4 or fewer nucleotide mismatches relative 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 comprising at least 3 or fewer nucleotide mismatches relative 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 comprising at least 2 or fewer nucleotide mismatches relative 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 comprising at least 1 or fewer nucleotide mismatches relative 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 comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0442] 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 that contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer mismatches, compared 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 contains at least 15 or fewer nucleotide mismatches relative 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 contains at least 10 or fewer nucleotide mismatches relative 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 contains at least 5 or fewer nucleotide mismatches relative 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 contains at least 4 or fewer nucleotide mismatches relative 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 contains at least 3 or fewer nucleotide mismatches relative 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 contains at least 2 or fewer nucleotide mismatches relative 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 contains at least 1 or fewer nucleotide mismatches relative 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 contains the codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0443] 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 that contains 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 compared 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 comprising at least 15 or fewer nucleotide mismatches relative 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 comprising at least 10 or fewer nucleotide mismatches relative 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 comprising at least 5 or fewer nucleotide mismatches relative 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 comprising at least 4 or fewer nucleotide mismatches relative 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 comprising at least 3 or fewer nucleotide mismatches relative 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 comprising at least 2 or fewer nucleotide mismatches relative 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 comprising at least 1 or fewer nucleotide mismatches relative 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 comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0444] 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 that contains 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 comprising at least 15 or fewer nucleotide mismatches relative 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 comprising at least 10 or fewer nucleotide mismatches relative 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 comprising at least 5 or fewer nucleotide mismatches relative 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 comprising at least 4 or fewer nucleotide mismatches relative 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 comprising at least 3 or fewer nucleotide mismatches relative 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 comprising at least 2 or fewer nucleotide mismatches relative 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 comprising at least 1 or fewer nucleotide mismatches relative 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 comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0445] 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 that contains 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mismatch, or fewer, relative 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 comprising at least 15 or fewer nucleotide mismatches relative 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 comprising at least 10 or fewer nucleotide mismatches relative 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 comprising at least 5 or fewer nucleotide mismatches relative 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 comprising at least 4 or fewer nucleotide mismatches relative 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 comprising at least 3 or fewer nucleotide mismatches relative 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 comprising at least 2 or fewer nucleotide mismatches relative 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 comprising at least 1 or fewer nucleotide mismatches relative 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 comprising the codon-optimized nucleic acid sequence of SEQ ID NO: 521.

[0446] Figure 14A shows 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 a fusion polypeptide), and a polyA sequence. Exemplary viral vector sequences (e.g., SEQ ID NOs: 520, 521, 534, and 535), as well as the nucleotides corresponding to the promoter, immunogen, and polyA sequences, are also shown in Figure 14A. In some embodiments, the CMV promoter sequence corresponding to nucleotides 594-1195 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 immunogen sequence corresponding to nucleotides 1256-4351 of SEQ ID NO: 520, or nucleotides 1256-4348 of SEQ ID NO: 521, or nucleotides 1256-2779 of SEQ ID NO: 534, or nucleotides 1256-3358 of SEQ ID NO: 535 is replaced with any of the immunogen sequences disclosed herein. In some embodiments, the immunogen sequences disclosed herein include any of the HIV immunogenic polypeptides or fusion proteins disclosed herein. In some embodiments, the polyA corresponding to nucleotides 4596-4619 of SEQ ID NO: 520, or nucleotides 4393-4616 of SEQ ID NO: 521, or nucleotides 2824-3047 of SEQ ID NO: 534, or nucleotides 3406-3629 of SEQ ID NO: 535 is replaced with another polyA sequence.

[0447] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 525 or 526. In some embodiments, the viral vector of 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 encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528.

[0448] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 524. In some embodiments, the viral vector of 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 encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528.

[0449] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 524, 525, or 526. In some embodiments, the viral vector of 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 any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528.

[0450] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 524, 525, or 526. In some embodiments, the viral vector of 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 encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 435, 527, and 528.

[0451] Figure 14B shows 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 a fusion polypeptide), and a polyA sequence. Exemplary viral vector sequences (e.g., SEQ ID NOs: 522, 523, and 537), as well as the nucleotides corresponding to the promoter, immunogen, and polyA sequences, are also shown in Figure 14B. In some embodiments, the 26S subgenomic promoter sequence corresponding to nucleotides 7513-7536 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 immunogen sequence corresponding to nucleotides 7571-10666 of SEQ ID NO: 522, or nucleotides 7571-10663 of SEQ ID NO: 523, or nucleotides 7571-9673 of SEQ ID NO: 538 is replaced with any of the immunogen sequences disclosed herein. In some embodiments, the immunogen sequences disclosed herein include any of the HIV immunogenic polypeptides or fusion proteins disclosed herein. In some embodiments, the polyA corresponding to nucleotides 10953-11072 of SEQ ID NO: 522, or nucleotides 10950-11069 of SEQ ID NO: 523, or nucleotides 9960-10039 of SEQ ID NO: 537 is replaced with another polyA sequence.

[0452] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 525 or 526. In some embodiments, the viral vector of 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 encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, 430 - 435, 527, and 528.

[0453] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 524. In some embodiments, the viral vector of 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 encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345 to 371, 373 to 377, 407 to 411, 422 to 423, 430 to 435, 527, and 528.

[0454] In some embodiments, the viral vector of 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, and the percent identity is over the full length of SEQ ID NO: 524, 525, or 526. In some embodiments, the viral vector of 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 any one of the amino acid sequences of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528, and the percent identity is over the full length of SEQ ID NOs: 345-371, 373-377, 407-411, 422-423, 430-435, 527, and 528.

[0455] In some embodiments, the expression vector, viral expression vector, or viral vector further comprises a polynucleotide encoding the cytokine or 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 is 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) Feb;60(2):261-71 (IL-2+IFN-α). In some embodiments, the vector (e.g., expression vector, viral expression vector, or viral vector) further comprises a non-coding immunostimulatory polynucleotide selected from pathogen-associated molecular patterns (PAMPs), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides, and immunostimulatory RNAs (isRNAs). Exemplary isRNAs include CV8102 (CureVac) and others described, for example, in WO 2016170176.

[0456] As described herein, there is further provided a host cell comprising one or more of the fusion polypeptides or one or more polynucleotides encoding one or more vectors that express the fusion polypeptide. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, e.g., E. coli. In another embodiment, the host cell is a eukaryotic cell, e.g., a yeast cell, a plant cell, an insect cell, a mammalian cell, e.g., a Chinese hamster ovary (CHO) system or a cell line derived from CHO (e.g., CHO-S, CHO DG44, ExpiCHO™, CHOZN® ZFN-modified GS- / - CHO cell line, CHO-K1, CHO-K1a), a COS cell, a BHK cell, an NSO cell, or a Bowes melanoma cell. Examples of human host cells include, inter alia, HeLa, 911, AT1080, A549, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™). Further, the fusion polypeptide can be expressed in yeast cells such as Pichia (e.g., Power et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces.

[0457] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny derived therefrom regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the originally transformed cell are included herein.

[0458] Optionally, as described herein, the host cell can be stably or transiently transfected with one or more polynucleotides encoding one or more fusion polypeptides. Optionally, the host cell can be infected with one or more vectors that express one or more fusion polypeptides, as described herein. In some embodiments, the host cell can be infected with and propagated by one or more replication-deficient or replication-competent vectors that express one or more fusion polypeptides, as described herein. Exemplary cells useful for infecting and / or propagating viral vectors include, but are not limited to, BHK-21, A549, Vero, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™) cells. In certain embodiments, the host cell expresses coxsackievirus and adenovirus receptor (CAR), e.g., MDCK, Caco-2, or Calu-3 host cells. In certain embodiments, the polynucleotide is integrated into the genome of the host cell. Lipid nanoparticles, polymer nanoparticles, and nanoemulsions

[0459] In certain embodiments, either the polynucleotide or the vector disclosed herein is 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 (LNP), polymer nanoparticles (PNP), and nanoemulsions. As used herein, "lipoplex" refers to cationic liposomes, which are non-viral (synthetic) lipid carriers of DNA.

[0460] As used herein, the term "lipid nanoparticle" refers to one or more spherical nanoparticles having an average diameter of about 10 to about 1000 nanometers and comprising a solid lipid core matrix capable of solubilizing lipophilic molecules. In certain embodiments, the lipid core is stabilized by a surfactant (e.g., an emulsifier) and can comprise one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate) comprising combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014, and U.S. Patent 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, a self-replicating or self-amplifying RNA molecule encoding one or more of the fusion polypeptides described herein is formulated or condensed in a polyethyleneimine (PEI)-polyplex delivery vehicle, which can be a nanoparticle, as described, for example, in Demoulins, et al., Nanomedicine. (2016) Apr;12(3):711-722, and Demoulins, et al., J Control Release. (2017) Nov 28;266:256-271. In some embodiments, the LNP comprises or is synthesized from ionizable lipids, phospholipids, cholesterol, PEGylated lipids, or any combination thereof. In some embodiments, the LNP comprises or is synthesized from the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0461] In some embodiments, 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.

[0462] 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), an oritenin-derived dendrimer, 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).

[0463] 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.

[0464] In various embodiments, the SAM, LNP, PNP, or nanoemulsion comprises one or more polynucleotides 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, or to any one of any one of SEQ ID NOs: 345-377, 407-411, 422-424, and 430-435.

[0465] 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 amino acid sequences of SEQ ID NO: 345 and 346; SEQ ID NO: 347 and 348; SEQ ID NO: 349 and 350; SEQ ID NO: 351 and 352; SEQ ID NO: 430 and 352; SEQ ID NO: 357 and 358; SEQ ID NO: 360 and 362; SEQ ID NO: 359 and 361; SEQ ID NO: 351 and 357; SEQ ID NO: 351 and 358; SEQ ID NO: 351 and 359; SEQ ID NO: 351 and 360; SEQ ID NO: 351 and 361; SEQ ID NO: 351 and 362; SEQ ID NO: 351 and 407; SEQ ID NO: 351 and 408; SEQ ID NO: 351 and 409; SEQ ID NO: 351 and 410; SEQ ID NO: 352 and 357; SEQ ID NO: 352 and 358; SEQ ID NO: 352 and 359; SEQ ID NO: 352 and 360; SEQ ID NO: 352 and 361; SEQ ID NO: 352 and 362; SEQ ID NO: 352 and 407; SEQ ID NO: 352 and 408; SEQ ID NO: 352 and 409; SEQ ID NO: 352 and 410; SEQ ID NO: 430 and 357; SEQ ID NO: 430 and 358; SEQ ID 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.

[0466] 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%, 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 that contains 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 compared 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 contains 15 or fewer amino acid mismatches relative 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 contains 10 or fewer amino acid mismatches relative 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 contains 5 or fewer amino acid mismatches relative 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 contains 4 or fewer amino acid mismatches relative 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 contains 3 or fewer amino acid mismatches relative 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 contains 2 or fewer amino acid mismatches relative 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 contains 1 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 527.

[0467] 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%, 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 that contains 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 compared 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 contains 15 or fewer amino acid mismatches relative 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 contains 10 or fewer amino acid mismatches relative 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 contains 5 or fewer amino acid mismatches relative 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 contains 4 or fewer amino acid mismatches relative 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 contains 3 or fewer amino acid mismatches relative 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 contains 2 or fewer amino acid mismatches relative 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 contains 1 or fewer amino acid mismatches relative to the amino acid sequence of SEQ ID NO: 528.

[0468] 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 any one of the amino acid sequences of SEQ ID NOs: 345 - 371, 373 - 377, 407 - 411, 422 - 423, and 430 - 435.

[0469] 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%, 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 that contains 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 compared to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches compared to the nucleic acid sequence of SEQ ID NO: 524.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 524. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 524.

[0470] 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%, 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 that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 525. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 525.

[0471] 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%, 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 that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 526. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 526.

[0472] 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%, 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 that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 522. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 522.

[0473] 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%, 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 that contains 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 relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523.In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 523. In some embodiments, the SAM, LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 523.

[0474] 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%, 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 contains 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 LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 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 that contains 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 that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 520. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 520.

[0475] 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%, 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 contains 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 relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 15 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 10 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521.In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 5 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 4 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 3 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 2 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains 1 or fewer nucleotide mismatches relative to the nucleic acid sequence of SEQ ID NO: 521. In some embodiments, the LNP, PNP, or nanoemulsion comprises a polynucleotide that contains the codon-optimized nucleic acid sequence of SEQ ID NO: 521. Composition

[0476] Provided are compositions such as pharmaceutical compositions or immunogenic compositions that comprise one or more of the fusion polypeptides described herein, or a polynucleotide encoding one or more of the fusion polypeptides described herein, or an expression vector, viral expression vector, or viral vector comprising one or more of such polynucleotides, or an LNP, PNP, or nanoemulsion comprising one or more of such polynucleotides. The composition may further comprise a pharmaceutically acceptable diluent, carrier, or excipient. Generally, the compositions described herein are immunogenic. In certain embodiments, the composition comprises a therapeutically effective amount of one or more of the fusion polypeptides described herein, or a polynucleotide encoding one or more of the fusion polypeptides described herein, or an expression vector, viral expression vector, or viral vector comprising one or more of such polynucleotides, or an LNP, PNP, or nanoemulsion comprising one or more of such polynucleotides.

[0477] Techniques for various pharmaceutically acceptable diluents, carriers, and excipients, as well as for the preparation and use of pharmaceutical compositions, are known to those skilled in the art in light of the present disclosure. Exemplary pharmaceutical compositions, as well as 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 Edition (2012), Pharmaceutical Press; Brunton, Knollman, and Hilal-Dandan, "Goodman and Gilman’s The Pharmacological Basis of Therapeutics," 13th Edition (2017), McGraw-Hill Education / Medical; McNally and Hastedt (Editors), "Protein Formulation and Delivery, 2nd Edition, 2007, CRC Press, Banga, "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems," 3rd Edition (2015), CRC Press; Lars Hovgaard, Frokjaer, and van de Weert (ed.), "Pharmaceutical Formulation Development of Peptides and Proteins," 2nd Edition (2012), CRC Press; Carpenter and Manning (ed.), "Rational Design of Stable Protein Formulations: Theory and Practice" (2002), Springer ("Pharmaceutical Biotechnology" (Volume 13)); Meyer (Editor), "Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, 2012, Woodhead Publishing.

[0478] 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, for example, 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, for example, intraoral, intranasal, or rectal 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, editors, "Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Biology)", 2019, Book 1937 in Methods in Molecular Biology Series, Humana Press, International Publication No. 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 formulation buffered with Tris and containing proline, lactose, and mannitol as stabilizing additives). Formulations of arenavirus vectors are described, for example, in International Publication Nos. 2009 / 083210, 2016 / 075250, and 2017 / 198726. In certain embodiments, the viral expression vector is delivered by, for example, microneedle-mediated delivery as described in Zaric, et al., Expert Opin Drug Deliv. (2017) Oct;14(10):1177-1187. Intranasal viral vaccination by administration of viral particles to the nasal cavity is described, for example, in Dorta-Estremera, et al., PLoS One. 2017 Dec 8;12(12):e0188807.Regarding rectal vaccination with virus particles administered to the rectum, for example, it is described in Patterson, et al., Clin Vaccine Immunol. (2012) May; 19(5): 629-37.

[0479] In some embodiments, each carrier, diluent, or excipient is "acceptable" in the sense that it is compatible with the other components of the pharmaceutical composition and not harmful to the subject. In many cases, a pharmaceutically acceptable carrier is an aqueous pH-buffered solution. Some examples of materials that can serve as pharmaceutically acceptable carriers, diluents, or excipients include the following: water; buffers, such as buffers having a pKa in the range of about 6.0 to about 8.0, such as physiologically acceptable buffers, such as buffers 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Hank's solution, Ringer's solution; ethyl alcohol; phosphate buffer; amino acids (e.g., charged amino acids including, but not limited to, aspartic acid, asparagine, glutamic acid, glutamine, histidine, arginine, lysine); and other non-toxic compatible su...

Claims

[Claim 1] The invention described in the present specification.

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