HIV vaccines and methods of making and using the same

Abstract

To provide HIV vaccines and methods of making and using the same.SOLUTION: Provided are: HIV-1 fusion polypeptides, polynucleotides encoding such fusion polypeptides, vectors expressing such fusion polypeptides, for use in eliciting an immune response against HIV-1; pharmaceutical and immunogenic compositions and kits comprising such fusion polypeptides, polynucleotides or vectors; and methods of use in treating and / or preventing HIV-1. Further provided are methods for design of antiviral vaccines, including vaccines to elicit an immune response against HIV-1.SELECTED DRAWING: None

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. 62 / 874,712, filed July 16, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on June 30, 2020 is named 1314_PF_SL.txt and is 449,156 bytes in size. [Background technology]

[0003] Human immunodeficiency virus type 1 (HIV-1) infection is a serious, life-threatening disease that remains one of the leading causes of morbidity and mortality worldwide, with approximately 36,900,000 infected individuals worldwide and 1,100,000 infected individuals in the United States (US) (National Center for HIV / AIDS Viral Hepatitis STD & TB Prevention: Division of HIV / AIDS Prevention, HIV in the United States and Dependent Areas. January 2019; UNAIDS, 2017 Global HIV Statistics. Fact Sheet - July 2018). Combination antiretroviral therapy (cART) for HIV-1 infection has significantly improved morbidity and mortality by suppressing viral replication, preserving immunological function, and avoiding progression to AIDS. However, despite cART, HIV-1 infection results in chronic immune activation and an increased risk of non-AIDS-related morbidity and mortality.

[0004] During the early stages of infection, HIV-1 integrates into the genome of memory cluster determinant 4 (CD4) T cells, a subset of which forms a long-lived reservoir of HIV-1-infected cells that persists despite treatment with antiretroviral therapy (ART) (Siliciano, et al., Nature Medicine (2003) 9(6):727-728). Eradication of the viral reservoir is a key component of an HIV cure strategy. Immune-based therapies may be an additional component of a combination approach to HIV cure or ART-free viral remission, which may include T cell and antibody-based vaccines, passive administration of antibodies, and immunomodulators.

[0005] The development of HIV T cell-specific vaccines has primarily focused on designing immunogens that provide universal coverage by addressing global HIV viral diversity. HIV-1 is defined by four groups (groups M, N, O, and P). Subtypes or clades (labeled A-K) within the M group and several cross-clade recombinant forms cause the majority of human disease. Strategies for designing vaccines that address the vast global viral sequence diversity include in silico-designed multivalent mosaic immunogens that capture common epitope variants within potential T cell epitopes (Fischer, et al., Nat Med, (2007) 13(1):100-6). These can be expressed as full-length artificial proteins or artificial recombinant proteins derived from regions with high sequence conservation (Ondondo, et al., Mol Ther, (2016) 24(4):832-42; Barouch, et al., Cell, (2013) 155(3):531-9). Subsequent iterations of in silico design algorithms led to the development of a computationally faster graph-based approach known as epigraphs (Theiler, et al., Sci Rep, (2016) 6:33987). These design approaches can be used to develop a single global vaccine or tailored to clades circulating within specific populations and regions. However, these approaches focus solely on viral diversity and do not consider the host genetic diversity that drives antigen presentation and T cell recognition, as well as the subsequent emergence of immune-driven escape variants.

[0006] Antigen-specific CD4+ and CD8+ T cells are associated with the control of viremia during acute infection and with slow disease progression and viremia control in individuals who maintain low viral loads in the absence of ART (elite controllers). Antigen-specific T cells recognize viral epitopes presented on MHC class I and II molecules. Human leukocyte antigen (HLA) class I alleles have been associated with HIV control in genome-wide association studies (GWAS) (Fellay, et al., Science, 2007. 317(5840):944-7; International, HIVCS, et al., Science, (2010) 330(6010):1551-7). These proteins present antigenic peptides from the sequence and induce effector and memory T cells. Current approaches to generating candidate HIV-1 vaccines focus on viral sequence diversity without adequately modeling the process of epitope generation across various host HLA alleles. This complex process of antigen presentation and T cell priming involves proteosomal cleavage, TAP transport, cross-presentation, MHC binding and peptide-MHC complex stability, and ultimately TCR recognition (Yewdell, et al. al., Nat Rev Immunol, (2003) 3(12):952-61). As a result, existing methods for generating T cell vaccines have met with limited success, for example, in some cases inducing an average of only four responses per patient (see, e.g., Priddy, et al., Clin Infect Dis (2008) 46(11):1769-81; Sekaly, et al., J Exp Med. (2008) 205(1):7-12, and Iaccino, et al., Retrovirology. (2008) 5:56).

[0007] Furthermore, highly mutated viruses such as HIV-1 pose unique challenges due to the high level of sequence diversity and the host immune response that drives some of that sequence diversity. The role of the adaptive immune response in driving diversity in HIV-1 is well documented, resulting in changes in viral sequences over time (Goulder, et al., Nature, (2001) 412(6844): 334-8; Kelleher, et al., J Exp Med, (2001) 193(3): 375-86; Schneidewind, et al., J Virol, (2007) 81(22): 12382-93; Kawashima, et al., Nature, (2009) 458(7238): 641-5; Leslie, et al., Nat Med, (2004) 10(3): 282-9; Phillips, et al., Nature, (1991) 354(6353): 453-9). Much of this diversity is driven by cytotoxic T lymphocytes that recognize peptide epitopes presented on MHC class I alleles. The selective pressure exerted by these T cell responses during chronic infection leads to the adaptation of HIV sequences. This sequence evolution drives HIV-1 diversity within individuals and across populations (Kawashima, et al., supra; Phillips, et al., supra). Furthermore, viral sequences undergo mutations that allow them to hide from host defenses. These sequences may resemble self-peptides or peptide sequences that induce central or peripheral tolerance. Standard vaccine design approaches may not be able to accommodate viral sequence variants and may result in inefficient use of vaccine capacity by including sequences that can induce responses that may cross-react with self-antigens. The impact of vaccines on human health cannot be overstated. Most of these are prophylactic vaccines, but they are effective in inducing conventional neutralizing antibodies against infectious disease targets. The development of therapeutic vaccines has made great strides in cancer immunotherapy, which has focused on developing vaccines that generate antigen-specific T cells. Many tumor-associated or tumor-specific antigens are self-antigens, necessitating vaccine design that overcomes immune tolerance. Recent innovations in identifying and predicting neoantigens arising from cancer-specific mutations provide promising targets that may not be subject to central or peripheral tolerance mechanisms. Various information strategies have been established to help identify neoantigens and predict their ability to induce strong T cell responses (see, for example, Bulik-Sullivan, et al., Nature Biotech (2019) 37:55-63). In the development of a therapeutic vaccine against HIV, antigenic targets are defined by the virus. Tools for predicting the effective presentation of viral sequences and their ability to stimulate an immune response are less well defined. This is relevant in the context of HIV, where high mutation rates coupled with host immune-mediated selective pressures result in the establishment of highly diverse quasispecies. Therefore, we developed informatics tools that allow the identification of conserved viral sequences in population-based consensus sequences or by individual deep sequencing of isolates, and can predict presentation, T cell priming, and HLA-driven escape pathways, which are useful in designing HIV vaccine immunogens. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] National Center for HIV / AIDS Viral Hepatitis STD & TB Prevention:Division of HIV / AIDS Prevent ion, HIV in the United States and Dependent Areas.January.2019;UNAIDS,2017 Global HIV Statistics.Fact Sheet-July.2018 [Non-patent document 2] Siliciano,et al.,Nature Medicine(2003)9(6):727-728 [Non-patent document 3] Fischer, et al., Nat Med, (2007) 13(1):100-6 [Non-patent document 4] Ondondo,et al.,Mol Ther,(2016)24(4):832-42 [Non-Patent Document 5] ;Barouch,et al.,Cell,(2013)155(3):531-9 [Non-patent document 6] Theiler,et al.,Sci Rep,(2016)6:33987 Summary of the Invention [Means for solving the problem]

[0009] At least the following embodiments are provided herein: Additional embodiments are described in the detailed embodiments and examples herein. Fusion Polypeptides

[0010] Embodiment 1: A fusion polypeptide comprising multiple polypeptide segments of one or more Human Immunodeficiency Virus-1 (HIV-1) proteins encoded by one or more HIV genes selected from Gag, Nef, Env, Pol, Rev, Tat, Rev, Vif, Vpr, and Vpu.

[0011] Embodiment 2: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 genes Env, Gag, Nef and Pol, e.g., excluding polypeptide segments encoded by HIV-1 Tat, Rev, Vif, Vpr and / or Vpu genes.

[0012] Embodiment 3: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 genes Gag, Nef and Pol, e.g., excluding polypeptide segments encoded by HIV-1 Env, Tat, Rev, Vif, Vpr and / or Vpu genes.

[0013] Embodiment 4: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 genes Gag and Nef, e.g., excluding polypeptide segments encoded by HIV-1 Env, Pol, Tat, Rev, Vif, Vpr and / or Vpu genes.

[0014] Embodiment 5: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 genes Pol and Nef, e.g., excluding polypeptide segments encoded by HIV-1 Env, Gag, Tat, Rev, Vif, Vpr and / or Vpu genes.

[0015] Embodiment 6: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 genes Pol and Env, e.g., excluding polypeptide segments encoded by HIV-1 Gag, Nef, Tat, Rev, Vif, Vpr and / or Vpu genes.

[0016] Embodiment 7: The fusion polypeptide of embodiment 1, wherein the plurality of polypeptide segments comprises or consists solely of polypeptide segments encoded by HIV-1 Pol genes, e.g., excluding polypeptide segments encoded by HIV-1 Env, Gag, Nef, Tat, Rev, Vif, Vpr and / or Vpu genes.

[0017] Embodiment 8: The fusion polypeptide of any one of embodiments 1 to 7, wherein the plurality of polypeptide segments does not contain segments encoded by one, two, three, or four of the HIV Tat, Rev, Vif, Vpr, and Vpu genes.

[0018] Embodiment 9: The fusion polypeptide of any one of embodiments 1 to 8, wherein the polypeptide segment is derived from a conserved region within a population of viral proteome sequences.

[0019] Embodiment 10: The fusion polypeptide of embodiment 9, wherein the conserved region is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% conserved between HIV-1 species in patient populations.

[0020] Embodiment 11: The fusion polypeptide of any one of embodiments 9 to 10, wherein the conserved region is conserved among one or more of HIV-1 Clades A to K, such as one or more of Clades A, B, C, D and G, or recombinant forms of one or more of HIV-1 Clades A to K, and combinations thereof.

[0021] Embodiment 12: The fusion polypeptide of any one of embodiments 1 to 11, comprising at least 5 and up to 40 polypeptide segments, such as 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, 40 polypeptide segments.

[0022] Embodiment 13: Each polypeptide segment is at least 8 amino acids in length and up to about 30, e.g., up to about 50, e.g., up to about 100, e.g., up to about 250 amino acids in length, e.g., at least 8 amino acids in length and up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 13. The fusion polypeptide of any one of embodiments 1 to 12, which is 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.

[0023] Embodiment 14: The total length of the fusion polypeptide is at least about 350 amino acids and up to about 1000 amino acids, e.g., 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, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 14. The fusion polypeptide of any one of embodiments 1 to 13, comprising 30, 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.

[0024] Embodiment 15: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises at least about 500 amino acids and at most about 1000 amino acids, such as at least about 500 amino acids and at most about 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.

[0025] Embodiment 16: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises or consists solely of polypeptide segments encoded by the HIV-1 genes Gag, Nef, and Pol, and is at least about 700 amino acids in length and at most about 800 amino acids in length, e.g., at least about 700 amino acids in length and at most about 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 amino acids in length (e.g., SEQ ID NOs: 345-350, 422-423 are exemplary fusion polypeptides).

[0026] Embodiment 17: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises or consists solely of polypeptide segments encoded by the HIV-1 genes Gag and Nef, including the optional N-terminal signal peptide, and is at least about 340 amino acids in length and at most about 500 amino acids in length, e.g., at least about 340 amino acids in length and at most about 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 amino acids in length (e.g., SEQ ID NOs: 351-356, 430 are exemplary fusion polypeptides).

[0027] Embodiment 18: The total length of the fusion polypeptide comprises or consists solely of polypeptide segments encoded by the HIV-1 genes Pol and Env, including the optional N-terminal signal peptide, and is at least about 335 amino acids in length and up to about 970 amino acids in length, for example, at least about 335 amino acids in length and up to about 340, 350, 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 or 970 amino acids in length (e.g., SEQ ID NOs: 357-366 are exemplary fusion polypeptides).

[0028] Embodiment 19: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises or consists solely of a polypeptide segment encoded by the HIV-1 gene Pol and is at least about 645 amino acids in length and at most about 675 amino acids in length, e.g., at least about 645 amino acids in length and at most about 650, 655, 660, 670, 675, or 680 amino acids in length (e.g., SEQ ID NOs: 407 to 410 are exemplary fusion polypeptides).

[0029] Embodiment 20: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises or consists solely of polypeptide segments encoded by the HIV-1 genes Env, Gag, Nef, and Pol, including the optional N-terminal signal peptide, and is at least about 360 amino acids in length and at most about 510 amino acids in length, e.g., at least about 360 amino acids in length and at most about 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, or 510 amino acids in length (e.g., SEQ ID NOs: 367-371, 424, 431-435 are exemplary fusion polypeptides).

[0030] Embodiment 21: The fusion polypeptide of any one of embodiments 1 to 13, wherein the total length of the fusion polypeptide comprises or consists solely of polypeptide segments encoded by the HIV-1 genes Env, Gag, Nef, and Pol, including the optional N-terminal signal peptide, and is at least about 760 amino acids in length and at most about 955 amino acids in length, for example, at least about 760 amino acids in length and at most about 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 955 amino acids in length (e.g., SEQ ID NOs: 373-377, 411 are exemplary fusion polypeptides).

[0031] Embodiment 22: The fusion polypeptide of any one of embodiments 1 to 14, wherein the total 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 or less.

[0032] Embodiment 23: A fusion polypeptide according to any one of embodiments 1 to 22, wherein each polypeptide segment comprises or consists of one or more predicted T-cell epitopes.

[0033] Embodiment 24: A fusion polypeptide according to any one of embodiments 1 to 23, comprising one or more polypeptide segments that bind to or are presented by one or more human HLA class I alleles (e.g., alleles of gene 1, 2, 3, 4, 5, or 6), e.g., within a single subject or among multiple patients.

[0034] Embodiment 25: At least one human HLA class I molecule, e.g., human A * 0201 25. A fusion polypeptide according to any one of embodiments 1 to 24, comprising one or more polypeptide segments that bind to or are presented by an HLA class I molecule.

[0035] Embodiment 26: A fusion polypeptide according to any one of embodiments 1 to 25, comprising one or more 8-mer, 9-mer, and / or 10-mer polypeptide segments presented by one or more human HLA class I alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., within a single subject.

[0036] Embodiment 27: The fusion polypeptide of any one of embodiments 1 to 25, comprising one or more 25mer polypeptide segments, each 25mer polypeptide segment comprising one or more 8mer, 9mer, and / or 10mer polypeptide segments presented by one or more human HLA class I alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., within a single subject.

[0037] Embodiment 28: A fusion polypeptide according to any one of embodiments 1 to 27, comprising one or more polypeptide segments that are intracellularly processed and presented by one or more human HLA class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., within a single subject.

[0038] Embodiment 29: A fusion polypeptide according to any one of embodiments 1 to 28, wherein one or more of the polypeptide segments abuts or is fused to an adjacent segment.

[0039] Embodiment 30: A fusion polypeptide according to any one of embodiments 1 to 28, wherein one or more of the polypeptide segments are linked to adjacent segments by one or more peptide linkers.

[0040] Embodiment 31: The fusion polypeptide of embodiment 30, wherein the 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.

[0041] Embodiment 32: The fusion polypeptide of embodiment 31, wherein the polyalanine linker comprises or consists of two or three consecutive alanine residues, e.g., AA, AAA (SEQ ID NO: 378), AAY (SEQ ID NO: 379) or AAX (SEQ ID NO: 380), and 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).

[0042] Embodiment 33: The fusion polypeptide of embodiment 31, wherein the flexible linker or polyglycine linker comprises or consists of GG, GGS (SEQ ID NO: 419), GSG (SEQ ID NO: 420) or GGGS (SEQ ID NO: 421).

[0043] Embodiment 34: The fusion polypeptide of embodiment 31, wherein 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 linking sequences, and combinations, derivatives or variants thereof.

[0044] Embodiment 35: The fusion polypeptide of embodiment 34, wherein 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).

[0045] Embodiment 36: The cleavable linker 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). 36. The fusion polypeptide of embodiment 34 or 35, comprising or consisting of an amino acid sequence, or comprising or consisting 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).

[0046] Embodiment 37: A fusion polypeptide according to embodiment 31, wherein the Nef linking 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), 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).

[0047] Embodiment 38: The fusion polypeptide of any one of embodiments 1 to 37, wherein the plurality of polypeptide segments comprises at least two polypeptide segments selected from SEQ ID NOs: 1 to 344, such as 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.

[0048] Embodiment 39: The fusion polypeptide of any one of embodiments 1 to 38, wherein the plurality of polypeptide segments comprises one or more segments of one or more viral proteins, or fragments or subsequences thereof, encoded by the HIV-1 Gag gene.

[0049] Embodiment 40: The fusion polypeptide of embodiment 39, wherein the one or more viral proteins encoded by the HIV-1 Gag gene are selected from p7, p17, and p24, and the fusion polypeptide does not contain any p6 protein.

[0050] Embodiment 41: The plurality of polypeptide segments comprises: SEQ ID NOs: 68 to 146 and 339 to 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 to 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 to 79 and 92 to 124, SEQ ID NOs: 70 to 71, 76 to 77 and 94 to 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 41. The fusion polypeptide of embodiment 39 or 40, comprising at least two polypeptide segments, such as 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 an amino acid sequence selected from SEQ ID NOs: 78 and 137.

[0051] Embodiment 42: The fusion polypeptide of any one of embodiments 39 to 41, wherein the plurality of polypeptide segments comprises at least two polypeptide segments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more segments, comprising or consisting of an HIV-1 Gag amino acid sequence corresponding to 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, wherein the amino acid positions are based on SEQ ID NO: 404.

[0052] Embodiment 43: The fusion polypeptide of any one of embodiments 39 to 42, wherein the plurality of polypeptide segments does not comprise one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 Gag amino acid sequence or subsequence thereof corresponding to amino acid residue positions selected from 1 to 30, 54 to 127, 138 to 146, 370 to 428, and 445 to 500, wherein the amino acid positions are based on SEQ ID NO: 404.

[0053] Embodiment 44: The fusion polypeptide of any one of embodiments 39 to 43, wherein the plurality of polypeptide segments does not comprise one, two, three, four, five or more polypeptide segments comprising or consisting of an HIV-1 Gag amino acid sequence of any one of SEQ ID NOs: 444 to 448, or a sequence or 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 SEQ ID NOs: 444 to 448.

[0054] Embodiment 45: The fusion polypeptide of any one of embodiments 1 to 44, wherein the plurality of polypeptide segments comprises one or more segments of a viral protein encoded by the HIV-1 Nef gene.

[0055] Embodiment 46: The plurality of polypeptide segments comprises: 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, Sequence numbers 149 to 152, SEQ ID NOs: 151 and 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 NOs: 153 and 165, and 46. ​​The fusion polypeptide of embodiment 45, comprising at least one polypeptide segment, such as 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, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 153.

[0056] Embodiment 47: The fusion polypeptide of embodiment 45 or 46, wherein the plurality of polypeptide segments comprises at least two polypeptide segments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more segments, comprising or consisting of an HIV-1 Nef amino acid sequence corresponding to 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, wherein the amino acid positions are relative to SEQ ID NO: 405.

[0057] Embodiment 48: The fusion polypeptide of any one of embodiments 45 to 47, wherein the plurality of polypeptide segments does not comprise one, two, three, or more polypeptide segments comprising or consisting of an HIV-1 Nef amino acid sequence or a subsequence thereof corresponding to amino acid residue positions selected from 1 to 63, 103 to 116, and 155 to 206, the amino acid positions being based on SEQ ID NO: 405.

[0058] Embodiment 49: The fusion polypeptide of any one of embodiments 45 to 48, wherein the plurality of polypeptide segments does not comprise one, two, three or more polypeptide segments comprising or consisting of the HIV-1 Nef amino acid sequence of any one of SEQ ID NOs: 449 to 451, or a sequence or 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 SEQ ID NOs: 449 to 451.

[0059] Embodiment 50: A fusion polypeptide according to any one of embodiments 1 to 49, wherein the plurality of polypeptide segments comprises or consists of one or more segments of viral proteins encoded by the HIV-1 Gag and Nef genes.

[0060] Embodiment 51: The plurality of polypeptide segments comprises: SEQ ID NOs: 68 to 79 and 92 to 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 51. The fusion polypeptide of embodiment 50, comprising at least two polypeptide segments, such as 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 an amino acid sequence selected from SEQ ID NOs: 71, 77, 95, 152 and 162.

[0061] Embodiment 52: In order from N-terminus to C-terminus, optionally linked or connected by one or more linkers: SEQ ID NOs: 70, 76, 94, 151 and 161, or A fusion polypeptide according to any one of embodiments 1 to 4 and 8 to 51, comprising or consisting of the polypeptide segments of SEQ ID NOs: 71, 77, 95, 152 and 162.

[0062] Embodiment 53: A fusion polypeptide according to any one of embodiments 1 to 52, wherein the plurality of polypeptide segments comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430.

[0063] Embodiment 54: A fusion polypeptide described in any one of embodiments 1, 2, 6 and 9 to 53, wherein the multiple polypeptide segments comprise one or more segments of one or more viral proteins encoded by the HIV-1 Env gene.

[0064] Embodiment 55: The fusion polypeptide of embodiment 54, wherein the one or more viral proteins encoded by the HIV-1 Env gene are selected from gp120 and gp41.

[0065] Embodiment 56: The plurality of polypeptide segments comprises: SEQ ID NOs: 1 to 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 to 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 56. The fusion polypeptide of embodiment 54 or 55, comprising at least two polypeptide segments, such as 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 an amino acid sequence selected from SEQ ID NOs: 6, 15 and 60.

[0066] Embodiment 57: The fusion polypeptide according to any one of embodiments 54 to 56, wherein the plurality of polypeptide segments comprises at least two polypeptide segments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more segments, comprising or consisting of an HIV-1 Env amino acid sequence corresponding to amino acid residue positions selected from 28 to 52, 34 to 48, 34 to 47, 36 to 44, 59 to 83, 64 to 83, 66 to 83, 67 to 75, 113 to 137, 235 to 259, 586 to 594, 586 to 610, 589 to 606 and 594 to 602, wherein the amino acid positions are based on SEQ ID NO: 403.

[0067] Embodiment 58: The fusion polypeptide of any one of embodiments 54 to 57, wherein the plurality of polypeptide segments does not comprise one, two, three, four, five, six, or more polypeptide segments comprising or consisting of an HIV-1 Env amino acid sequence or subsequence thereof corresponding to amino acid residue positions selected from 1 to 27, 53 to 58, 84 to 112, 138 to 234, 269 to 474, 490 to 501, 611 to 856, wherein the amino acid positions are based on SEQ ID NO: 403.

[0068] Embodiment 59: The fusion polypeptide of any one of embodiments 54 to 57, wherein the plurality of polypeptide segments does not comprise one, two, three, four, five or more polypeptide segments comprising or consisting of an HIV-1 Env amino acid sequence of any one of SEQ ID NOs: 437 to 443, or a sequence or 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 SEQ ID NOs: 437 to 443.

[0069] Embodiment 60: A fusion polypeptide according to any one of embodiments 1 to 58, wherein the plurality of polypeptide segments comprises or consists of one or more segments of one or more viral proteins encoded by HIV-1 Pol genes.

[0070] Embodiment 61: The fusion polypeptide of embodiment 60, wherein the one or more viral proteins encoded by the HIV-1 Pol gene are selected from one or more of protease (PR), reverse transcriptase (RT), and integrase (INT).

[0071] Embodiment 62: The plurality of polypeptide segments comprises: SEQ ID NOs: 174 to 337 and 343 to 344, Sequence numbers 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, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 7, 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, Sequence 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, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287 6, 237, 238, 239, 240, 241, 242, 243, 244, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 282, 283, 294, 295, 296, 297, 298, 299, 300, 301, 302, 305, 306, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 and 337, SEQ ID NOs: 176, 177, 188, 189, 213, 214, 223, 224, 259, 260, 282, 283, 294, 295, 305, 306, 319 and 320, SEQ ID NOs: 180, 181, 186, 187, 221, 222, 294, 295, 321 and 322, SEQ ID NOs: 182 to 202, 292 to 302, 305 and 306, SEQ ID NOs: 188, 189, 294, 295, 305 and 306, Sequence 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, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317 1, 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, SEQ ID NOs: 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319 and 320, SEQ ID NOs: 192, 201, 202, 215, 216, 217, 218, 219, 220, 229, 230, 231, 240, 241, 242, 243, 244, 265, 276, 277, 298, 299, 302, 311, 312, 327, 328, 331, 332, 333, 336 and 337, SEQ ID NOs: 192, 201, 215, 217, 219, 229, 230, 240, 241, 243, 265, 276, 298, 302, 311, 327, 331, 333 and 336, Sequence 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 2, 274, 275, 276, 277, 296, 297, 298, 299, 300, 301, 302, 303, 304, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 343, 344, 375 and 376, SEQ ID NOs: 190, 197, 209, 210, 211, 225, 227, 234, 238, 261, 296, 300, 303, 323, 325, 329 and 334, and 62. The fusion polypeptide of embodiment 60 or 61, comprising at least two polypeptide segments, such as 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 an amino acid sequence selected from SEQ ID NOs: 192, 215, 217, 219, 229, 230, 276, 298, 302, 327, 331, 333 and 336.

[0072] Embodiment 63: The plurality of polypeptide segments comprises: Sequence numbers 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, 1 81, 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, 28 2, 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 to 56, 182 to 202, 292 to 302, 305 and 306, SEQ ID NOs: 28, 29, 41, 42, 188, 189, 294, 295, 305 and 306, Sequence numbers 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 63. The fusion polypeptide according to any one of embodiments 54 to 62, comprising at least two polypeptide segments, such as 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 an amino acid sequence selected from SEQ ID NOs: 4, 5, 11, 12, 37, 38, 176, 177, 213, 214, 223, 224, 259, 260, 282, 283, 319 and 320.

[0073] Embodiment 64: In order from N-terminus to C-terminus, optionally linked or connected by one or more linkers: SEQ ID NOs: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 188, 305, 28, 41 and 294, SEQ ID NOs: 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 189, 306, 29, 42 and 295, SEQ ID NOs: 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 305, 319, 259, 282, 223, 213, 294, 176 and 188, SEQ ID NOs: 306, 320, 260, 283, 224, 214, 295, 177 and 189, SEQ ID NOs: 305, 294, 223, 213, 176, 259, 319, 188 and 282, SEQ ID NOs: 306, 295, 224, 214, 177, 260, 320, 189 and 283, SEQ ID NOs: 305, 294, 319, 259, 282, 223, 176 and 188, SEQ ID NOs: 306, 295, 320, 260, 283, 224, 177 and 189, SEQ ID NOs: 305, 223, 294, 176, 259, 319, 188 and 282, or The fusion polypeptide according to any one of embodiments 1, 6, 9 to 38 and 54 to 63, comprising or consisting of the polypeptide segments of SEQ ID NOs: 306, 224, 295, 177, 260, 320, 189 and 283.

[0074] Embodiment 65: The plurality of polypeptide segments is selected from the group consisting of 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, 406-44 HIV-1 corresponding to amino acid residue positions selected from 30, 553 to 577, 642 to 666, 650 to 658, 759 to 783, 767 to 775, 768 to 792, 776 to 784, 834 to 858, 940 to 964, 947 to 971, 948 to 956, 948 to 972, 955 to 963, 956 to 964, 980 to 1003, and 988 to 996 65. The fusion polypeptide of any one of embodiments 60 to 64, comprising at least two polypeptide segments, 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, 39, 30 or more segments, comprising or consisting of the Pol amino acid sequence, wherein the amino acid positions are based on SEQ ID NO: 406.

[0075] Embodiment 66: The fusion polypeptide of any one of embodiments 60 to 65, wherein the plurality of polypeptide segments does not include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polypeptide segments comprising or consisting of an HIV-1 Pol amino acid sequence or subsequence thereof corresponding to amino acid residue positions selected from 1 to 55, 118 to 128, 321 to 325, 355 to 366, 432 to 541, 607 to 641, 667 to 682, 709 to 746, 828 to 833, and 921 to 930, wherein the amino acid positions are based on SEQ ID NO: 406.

[0076] Embodiment 67: The fusion polypeptide of any one of embodiments 60 to 66, wherein the plurality of polypeptide segments does not comprise one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 Pol amino acid sequence of any one of SEQ ID NOs: 452 to 461, or a sequence or 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 SEQ ID NOs: 452 to 461.

[0077] Embodiment 68: A fusion polypeptide according to any one of embodiments 1, 6 to 38 and 54 to 67, wherein the plurality of polypeptide segments comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410.

[0078] Embodiment 69: A fusion polypeptide according to any one of embodiments 1, 3 and 8 to 68, wherein the plurality of polypeptide segments comprises or consists of segments of viral proteins encoded by the Gag, Nef and Pol genes.

[0079] Embodiment 70: The plurality of polypeptide segments comprises: Sequence numbers 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 70, 31, 32, 34, 35, 36, 37, 38, 39, 40 or more segments comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 76, 77, 86, 87, 94, 95, 151, 152, 181, 182, 186, 187, 221, 222, 294, 195, 307, 308, 321, 322.

[0080] Embodiment 71: In order from N-terminus to C-terminus, optionally linked or connected by one or more linkers: SEQ ID NOs: 76, 86, 94, 180, 186, 221, 294, 307, 321 and 151, or The fusion polypeptide of embodiment 69 or 70, comprising or consisting of the polypeptide segments of SEQ ID NOs: 77, 87, 95, 181, 187, 222, 295, 308, 322 and 152.

[0081] Embodiment 72: A fusion polypeptide according to any one of embodiments 69 to 71, wherein the plurality of polypeptide segments comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 345 to 350, the sequences in Table 1, and SEQ ID NOs: 422 to 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 to 350, the sequences in Table 1, and SEQ ID NOs: 422 to 424.

[0082] Embodiment 73: The plurality of polypeptide segments comprises or consists of segments of viral proteins encoded by Gag, Pol, Env, and Nef genes, and each of the plurality of polypeptide segments is a human HLA allele A. * 73. The fusion polypeptide of any one of embodiments 1 to 72, which is capable of binding to or being presented by O201.

[0083] Embodiment 74: The fusion polypeptide of embodiment 73, wherein each of the plurality of polypeptide segments is 8 to 35 amino acids in length, such as 9 to 34 amino acids in length, such as 9 to 25 amino acids in length.

[0084] Embodiment 75: The plurality of polypeptide segments comprises: 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, Sequence numbers 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, 1 92, 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 amino acid sequences comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 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; 75. The fusion polypeptide of embodiment 73 or 74, comprising 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.

[0085] Embodiment 76: In order from N-terminus to C-terminus, optionally linked or connected by one or more linkers: Sequence numbers 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 NOs: 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 76. The fusion polypeptide according to any one of embodiments 73 to 75, comprising or consisting of the polypeptide segments of SEQ ID NOs: 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.

[0086] Embodiment 77: A fusion polypeptide according to any one of embodiments 73 to 76, wherein the plurality of polypeptide segments comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 367 to 377, 411, 431 to 435, or a sequence which 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 to 377, 411, 431 to 435.

[0087] Embodiment 78: The fusion polypeptide of any one of embodiments 1 to 77, wherein the fusion polypeptide does not comprise the amino acid sequence YMDD (SEQ ID NO: 462) or YVDD (SEQ ID NO: 463).

[0088] Embodiment 79: The fusion polypeptide of embodiment 78, wherein the fusion polypeptide does not comprise one or more amino acid sequences selected from SEQ ID NOs: 215, 216, 217, 218, 219 and 220.

[0089] Embodiment 80: The fusion polypeptide of embodiment 78 or 79, wherein the fusion polypeptide does not comprise one or more amino acid sequences selected from SEQ ID NOs: 209, 210, 211, 212, 213, 214, 343 and 344.

[0090] Embodiment 81: A fusion polypeptide comprising the amino acid sequence of SEQ ID NOs: 345-352, 357-362, 367, 373, 407-411 or 422-424, or a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 345-352, 357-362, 367, 373, 407-411, 422-424 and 431-435.

[0091] Embodiment 82: A fusion polypeptide according to any one of embodiments 1 to 81, comprising an N-terminal signal peptide or leader sequence.

[0092] Embodiment 83: The fusion polypeptide of embodiment 82, wherein the signal peptide or leader sequence is derived from a source protein selected from a serum protein, a cytokine, a chemokine, a chaperone protein, an invariant protein, and a protein that targets proteins to the lysosomal compartment.

[0093] Embodiment 84: The fusion polypeptide of embodiment 82 or 83, wherein the signal peptide or leader sequence is derived from a source protein selected from the group consisting of 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).

[0094] Embodiment 85: A fusion polypeptide according to any one of embodiments 82 to 84, wherein the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs: 393 to 402 and 412 to 413, or a sequence which is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 393 to 402 and 412 to 413.

[0095] Embodiment 86: The fusion polypeptide of any one of embodiments 1 to 85, wherein the fusion polypeptide is recombinantly produced or chemically synthesized.

[0096] Embodiment 87: The fusion polypeptide of any one of embodiments 1 to 86, wherein the fusion polypeptide is capable of inducing, enhancing or stimulating an immune response in humans.

[0097] Embodiment 88: The fusion polypeptide of any one of embodiments 1 to 87, wherein the fusion polypeptide is capable of inducing, enhancing or stimulating an immune response against HIV-1 in humans.

[0098] Embodiment 89: A fusion polypeptide according to any one of embodiments 1 to 88, wherein the fusion polypeptide is capable of inducing, promoting or stimulating the proliferation and / or activation of one or more cell types selected from monocyte-derived dendritic cells (DCs), CD8+ T cells and CD4+ T cells. Polynucleotides, lipoplexes, expression cassettes, vectors, host cells

[0099] Embodiment 90: A polynucleotide encoding one or more fusion polypeptides according to any one of embodiments 1 to 89.

[0100] Embodiment 91: The polynucleotide of embodiment 90, wherein the polynucleotide comprises or is in the form of cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA).

[0101] Embodiment 92: The polynucleotide of embodiment 91, wherein the polynucleotide comprises one or more self-replicating or self-amplifying alphavirus replicons.

[0102] Embodiment 93: A polynucleotide according to any one of embodiments 90 to 92, comprising the nucleic acid sequence of any one of SEQ ID NOs: 414 to 418, or a nucleic acid sequence which 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: 414 to 418.

[0103] Embodiment 94: A lipoplex, such as a lipid nanoparticle (LNP), comprising a polynucleotide according to any one of embodiments 90 to 93.

[0104] Embodiment 95: An expression cassette comprising the polynucleotide of any one of embodiments 90 to 93 operably linked to one or more regulatory sequences.

[0105] Embodiment 96: The expression cassette of embodiment 95, wherein the polynucleotide is operably linked to and under the control of a constitutive promoter.

[0106] Embodiment 97: An expression cassette according to embodiment 95 or 96, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, and an EF1a promoter.

[0107] Embodiment 98: A vector comprising one or more polynucleotides according to any one of embodiments 90 to 93 or an expression cassette according to any one of embodiments 95 to 97.

[0108] Embodiment 99: The vector of embodiment 98, wherein the vector is a plasmid vector, a bacterial vector, or a viral vector.

[0109] Embodiment 100: The vector of embodiment 98 or 99, wherein the vector is a viral vector or a viral expression vector.

[0110] Embodiment 101: The vector according to any one of embodiments 98 to 100, wherein the viral vector or viral expression vector is derived from a DNA virus or an RNA virus.

[0111] Embodiment 102: The vector of any one of embodiments 98 to 101, wherein the viral vector or 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.

[0112] Embodiment 103: The vector of any one of embodiments 98 to 102, wherein the viral vector or 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)), Paramyxoviridae (e.g., measles virus), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Marabavesiculovirus), Togaviridae (e.g., alphavirus).

[0113] Embodiment 104: The vector of any one of embodiments 98 to 103, wherein the viral vector or viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Kali 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).

[0114] Embodiment 105: The vector of embodiment 104, wherein the viral vector or viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus).

[0115] Embodiment 106: The vector of any one of embodiments 98 to 103, wherein the viral vector or viral expression vector is a human adenovirus or a simian adenovirus (e.g., a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus adenovirus).

[0116] Embodiment 107: The viral vector or viral expression vector is selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (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), ChA d22(AdC22), ChAd24(AdC24), ChAdY25, ChAd26(AdC26), ChAd28(AdC28), ChAd30(AdC30), ChAd31(AdC31), ChAd37(AdC3 7), ChAd38(AdC38), ChAd43(AdC43), ChAd44(AdC44), ChAd55(AdC55), ChAd63(AdC63), ChAdV63, ChAd68(AdC68), ChAd7 3(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).

[0117] Embodiment 108: The vector of any one of embodiments 98 to 107, wherein the viral vector or viral expression vector is replication-defective, replication-deficient, replication-attenuated or replication-competent.

[0118] Embodiment 109: The vector according to any one of embodiments 98 to 108, wherein the viral vector or viral expression vector is an adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, 430 to 435, 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 to 377, 407 to 411, 422 to 424, 430 to 435.

[0119] Embodiment 110: The vector is selected from the group consisting of SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407 109. The vector of any one of embodiments 98-109, comprising 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 sequence of SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or SEQ ID NOs: 409 and 410.

[0120] Embodiment 111: A host cell comprising one or more polynucleotides according to any one of embodiments 90 to 93 or one or more vectors according to any one of embodiments 98 to 110.

[0121] Embodiment 112: The host cell of embodiment 111, wherein the one or more polynucleotides are not integrated into the host cell genome, e.g., are episomal.

[0122] Embodiment 113: The host cell of embodiment 111, wherein the one or more polynucleotides are integrated into the host cell genome.

[0123] Embodiment 114: The host cell according to any one of embodiments 111 to 113, wherein the host cell is a mammalian cell, for example a human cell, for example a cell line selected from BHK-21, A549, Vero, HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™) cells, MDCK, Caco-2 and Calu-3.

[0124] Embodiment 115: The host cell according to any one of embodiments 111 to 114, wherein the host cell is in vitro.

[0125] Embodiment 116: The host cell of any one of embodiments 111 to 114, wherein the host cell is in vivo. composition

[0126] Embodiment 117: An immunogenic composition comprising one or more of the fusion polypeptides according to any one of embodiments 1 to 89, or one or more polynucleotides according to any one of embodiments 90 to 93, or one or more vectors according to any one of embodiments 98 to 110, and a pharmaceutically acceptable carrier.

[0127] Embodiment 118: An immunogenic composition according to embodiment 117, comprising two or more of the fusion polypeptides according to any one of embodiments 1 to 89, or two or more polynucleotides according to any one of embodiments 90 to 93, or two or more vectors according to any one of embodiments 98 to 110.

[0128] Embodiment 119: An immunogenic composition according to any one of embodiments 117 to 118, wherein the one or more polynucleotides comprise or are in the form of DNA, cDNA, mRNA, or self-replicating RNA.

[0129] Embodiment 120: 1) in order from N-terminus to C-terminus, optionally linked or connected by one or more linkers; SEQ ID NOs: 70, 76, 94, 151 and 161, or one or more fusion polypeptides comprising or consisting of the polypeptide segments of SEQ ID NOs: 71, 77, 95, 152 and 162; 2) in N-terminal to C-terminal order, optionally linked or connected by one or more linkers; SEQ ID NOs: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 188, 305, 28, 41 and 294, SEQ ID NOs: 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 189, 306, 29, 42 and 295, SEQ ID NOs: 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 305, 319, 259, 282, 223, 213, 294, 176 and 188, SEQ ID NOs: 306, 320, 260, 283, 224, 214, 295, 177 and 189, SEQ ID NOs: 305, 294, 223, 213, 176, 259, 319, 188 and 282, SEQ ID NOs: 306, 295, 224, 214, 177, 260, 320, 189 and 283, SEQ ID NOs: 305, 294, 319, 259, 282, 223, 176 and 188, SEQ ID NOs: 306, 295, 320, 260, 283, 224, 177 and 189, SEQ ID NOs: 305, 223, 294, 176, 259, 319, 188 and 282, or and one or more fusion polypeptides comprising or consisting of the polypeptide segments of SEQ ID NOs: 306, 224, 295, 177, 260, 320, 189 and 283.

[0130] Embodiment 121: The immunogenic composition according to any one of embodiments 117 to 120, comprising one or more adenoviral vectors, each adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, 430 to 435, 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 to 377, 407 to 411, 422 to 424, 430 to 435.

[0131] Embodiment 122: The method of claim 1, further comprising administering to a patient a viral vector selected from the group consisting of SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 363, SEQ ID NOs: 352 and 364, SEQ ID NOs: 352 and 365, SEQ SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or SEQ ID NO: 409 and 410. The immunogenic composition of any one of embodiments 117-121, comprising one 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 sequence of

[0132] Embodiment 123: 1) one or more fusion polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430; 2) one or more fusion polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410.

[0133] Embodiment 124: A pharmaceutical composition comprising one or more of the fusion polypeptides according to any one of embodiments 1 to 89, or one or more polynucleotides according to any one of embodiments 90 to 93, or one or more vectors according to any one of embodiments 98 to 110, and a pharmaceutically acceptable carrier.

[0134] Embodiment 125: The pharmaceutical composition of embodiment 124, comprising two or more fusion polypeptides, two or more polynucleotides, or two or more vectors.

[0135] Embodiment 126: The pharmaceutical composition of embodiment 124 or 125, further comprising one or more of an adjuvant, an immunostimulant, a detergent, a micelle-forming agent, and an oil.

[0136] Embodiment 127: The pharmaceutical composition of embodiment 126, wherein the immunomodulatory agent is selected from a toll-like receptor (TLR) agonist, a cytokine (e.g., 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), such as pathogen-activating molecular patterns (PAMPs), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides, and immunostimulatory RNA (isRNA, e.g., CV8102)) inhibitors of inhibitory immune checkpoint proteins or stimulators of stimulatory immune checkpoint proteins.

[0137] Embodiment 128: The pharmaceutical composition of any one of embodiments 124 to 127, which is formulated for administration via a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., oral, nasal, rectal, vaginal).

[0138] Embodiment 129: The pharmaceutical composition of any one of embodiments 124 to 128, which is formulated as a liquid.

[0139] Embodiment 130: A pharmaceutical composition according to any one of embodiments 124 to 128, wherein the composition is lyophilized. kit

[0140] Embodiment 131: A kit comprising one or more unit doses of one or more of the fusion polypeptides according to any one of embodiments 1 to 89, or one or more polynucleotides according to any one of embodiments 90 to 93, or one or more vectors according to any one of embodiments 98 to 110, or one or more immunogenic compositions according to any one of embodiments 117 to 121, or one or more pharmaceutical compositions according to any one of embodiments 124 to 130.

[0141] Embodiment 132: The kit of embodiment 131, wherein the one or more unit doses are in a single container.

[0142] Embodiment 133: The kit of embodiment 131, wherein the one or more unit doses are in two or more separate containers.

[0143] Embodiment 134: A kit according to any one of embodiments 131 to 133, comprising one or more containers selected from the group consisting of vials, ampoules, and pre-loaded syringes.

[0144] Embodiment 135: A kit according to any one of embodiments 131 to 134, comprising one or more containers containing one or more fusion polypeptides, one or more polynucleotides, or one or more vectors in aqueous solution.

[0145] Embodiment 136: A kit according to any one of embodiments 131 to 135, wherein one or more unit doses are the same.

[0146] Embodiment 137: A kit according to any one of embodiments 131 to 135, wherein one or more unit doses are different.

[0147] Embodiment 138: A method for the treatment of a patient comprising administering to the patient one or more unit doses of one or more viral vectors according to any one of embodiments 98 to 110, wherein the unit dose is about 10 3 ~about 10 15 Viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (vp), for example, about 10 4 ~about 10 7 Viral FFU or PFU or IU or vp, for example, about 10 3 ~about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 138. The kit of any one of embodiments 131 to 137, wherein the amount of virus is in the range of FFU or PFU or IU or vp.

[0148] Embodiment 139: A kit according to any one of embodiments 131 to 138, comprising two or more of the fusion polypeptides according to any one of embodiments 1 to 89, or two or more polynucleotides according to any one of embodiments 90 to 93, or two or more vectors according to any one of embodiments 98 to 110.

[0149] Embodiment 140: The method comprises the steps of: (a) synthesizing a fusion polypeptide comprising: (i) synthesizing a fusion polypeptide comprising: (a) synthesizing a fusion polypeptide comprising: 1) in order from N-terminus to C-terminus, optionally linked or connected by one or more linkers; SEQ ID NOs: 70, 76, 94, 151 and 161, or one or more fusion polypeptides comprising or consisting of the polypeptide segments of SEQ ID NOs: 71, 77, 95, 152 and 162; 2) in N-terminal to C-terminal order, optionally linked or connected by one or more linkers; SEQ ID NOs: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 188, 305, 28, 41 and 294, SEQ ID NOs: 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 189, 306, 29, 42 and 295, SEQ ID NOs: 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 305, 319, 259, 282, 223, 213, 294, 176 and 188, SEQ ID NOs: 306, 320, 260, 283, 224, 214, 295, 177 and 189, SEQ ID NOs: 305, 294, 223, 213, 176, 259, 319, 188 and 282, SEQ ID NOs: 306, 295, 224, 214, 177, 260, 320, 189 and 283, SEQ ID NOs: 305, 294, 319, 259, 282, 223, 176 and 188, SEQ ID NOs: 306, 295, 320, 260, 283, 224, 177 and 189, SEQ ID NOs: 305, 223, 294, 176, 259, 319, 188 and 282, or and one or more fusion polypeptides comprising or consisting of the polypeptide segments of SEQ ID NOs: 306, 224, 295, 177, 260, 320, 189 and 283.

[0150] Embodiment 141: The method comprises the steps of: (a) synthesizing a fusion polypeptide comprising: (a) synthesizing a fusion polypeptide comprising: (i) synthesizing a fusion polypeptide comprising: 1) one or more fusion polypeptides comprising one or more polynucleotides encoding the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430, or one or more vectors capable of expressing the same; 2) one or more fusion polypeptides comprising one or more polynucleotides encoding the amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410, or one or more fusion polypeptides comprising one or more vectors capable of expressing the same.

[0151] Embodiment 142: The method of claim 142, comprising one or more polynucleotides encoding fusion polypeptides or one or more vectors expressing fusion polypeptides, wherein the fusion polypeptides are linked or connected in N-terminal to C-terminal order, optionally by one or more linkers. Sequence numbers 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 NOs: 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 142. The kit according to any one of embodiments 131 to 141, comprising or consisting of the polypeptide segments of SEQ ID NOs: 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.

[0152] Embodiment 143: A kit according to any one of embodiments 131 to 142, comprising one or more polynucleotides encoding a fusion polypeptide or one or more vectors expressing a fusion polypeptide, wherein the fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 411, 422 to 424 and 430 to 435, or a sequence which 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 to 377, 411, 422 to 424 and 430 to 435.

[0153] Embodiment 144: A kit according to any one of embodiments 131 to 143, comprising one or more adenoviral vectors, each adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, 430 to 435, 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 to 377, 407 to 411, 422 to 424, 430 to 435.

[0154] Embodiment 145: The method comprises one or more viral vectors, each viral vector comprising: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, Sequence numbers 352 and 407, sequence numbers 352 and 408, sequence numbers 352 and 409, sequence numbers 352 and 410, sequence numbers 430 and 357, sequence numbers 430 and 358, sequence numbers 430 and 359, sequence numbers 430 and 360, sequence numbers 430 and 361, sequence numbers 430 and 362, sequence numbers 407 and 409, sequence numbers 407 and 408, sequence numbers 408 and 410, or sequence numbers 145. The kit of any one of embodiments 131 to 144, comprising 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 409 and 410.

[0155] Embodiment 146: The kit of any one of embodiments 131 to 145, further comprising one or more unit doses of one or more additional therapeutic agents.

[0156] Embodiment 147: The kit of embodiment 146, comprising one or more agents that activate latent HIV, such as one or more latent reactivating agents (LRAs).

[0157] Embodiment 148: The kit of embodiment 146 or 147, comprising one or more LRAs selected from the group consisting of one or more agonists or activators of Toll-like receptors (TLRs), histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitors of apoptosis proteins (IAP) antagonists, and a second mitochondria-derived activator of caspases (SMAC) mimetic.

[0158] Embodiment 149: A kit according to any one of embodiments 146 to 148, comprising one or more agonists or activators of one or more Toll-like receptors (TLRs).

[0159] Embodiment 150: The kit of embodiment 149, wherein the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0160] Embodiment 151: The kit of embodiment 149 or 150, wherein the TLR7 agonist is selected from the group consisting of GS9620 (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).

[0161] Embodiment 152: The kit according to any one of embodiments 149 to 151, wherein the TLR9 agonist is selected from the group consisting of AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, ritenimod, 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.

[0162] Embodiment 153: A kit according to any one of embodiments 146 to 152, comprising one or more interleukin receptor agonists of interleukins selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, GM-CSF, FLT3LG.

[0163] Embodiment 154: The kit of embodiment 153, comprising one or more cytokines selected from the group consisting of 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.

[0164] Embodiment 155: A kit according to any one of embodiments 146 to 154, comprising one or more innate immune activators.

[0165] Embodiment 156: The kit of embodiment 155, wherein the one or more innate immune activators comprise a non-coding immunostimulatory polynucleotide (e.g., pathogen-activating molecular pattern (PAMP), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide, and immunostimulatory RNA (isRNA, e.g., CV8102)), an agonist of a receptor selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain-containing 2 (NOD2).

[0166] Embodiment 157: A kit according to any one of embodiments 146 to 156, comprising one or more blockers, antagonists, or inhibitors of inhibitory immune checkpoint proteins or receptors, and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors.

[0167] Embodiment 158: The one or more immune checkpoint proteins or receptors are selected from the group consisting of CD27, CD70, CD40, CD40LG, CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4), CD276 (B7H3), V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), immunoglobulin superfamily member 11 (IGSF11, VSIG3), natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6), and HERV-H. Long-Term Receptor Associated 2 (HHLA2, B7H7), Inducible T Cell Costimulator (ICOS, CD278), Inducible T Cell Costimulator Ligand (ICOSLG, B7H2), TNF Receptor Superfamily Member 4 (TNFRSF4, OX40), TNF Superfamily Member 4 (TNFSF4, OX40L), TNFRSF8 (CD30), TNFSF8 (CD30L), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL), TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML), and CD272 (B and T Lymphocyte-Associated (BTLA) ), TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF), TNFRSF18 (GITR), TNFSF18 (GITRL), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), CD274 (CD274, PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), CD80 (B7-1), CD28, nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155), PVR-related immunoglobulin domain-containing (PVRIG, CD112R),T cell immunoreceptor containing Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), galectin 9 (LGALS9), lymphocyte activation 3 (LAG3, CD223), signaling lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150), lymphocyte antigen 9 (LY9, CD229, SLAMF3), and SLAM family member 6 (SLAM F6, CD352), SLAM family member 7 (SLAMF7, CD319), UL16 binding protein 1 (ULBP1), UL16 binding protein 2 (ULBP2), UL16 binding protein 3 (ULBP3), retinoic acid early transcript 1E (RAET1E; ULBP4), retinoic acid early transcript 1G (RAET1G; ULBP5), retinoic acid early transcript 1L (RAET1L; ULBP6), lymphocyte antigen 3 (CD223), killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1), killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C), killer cell lectin-like receptor C3 (KLRC3, NKG2E), killer cell lectin-like receptor C4 (KLRC4, NKG2F), and killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1), killer cell lectin-like receptor D1 (KLRD1), and SLAM family member 7 (SLAMF7).

[0168] Embodiment 159: The kit of embodiment 157 or 158, comprising one or more blockers, antagonists, or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors.

[0169] Embodiment 160: The T cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of CD274 (CD274, PDL1, PD-L1), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), programmed cell death 1 (PDCD1, PD1, PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), CD276 (B7H3), and 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), and Ig and IT 160. The kit of embodiment 159, wherein the immunoreceptor is selected from the group consisting of a T-cell immunoreceptor comprising an IM domain (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 a long cytoplasmic tail 1 (KIR, CD158E1), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), and killer-cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1).

[0170] Embodiment 161: A kit according to any one of embodiments 157 to 160, comprising one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors.

[0171] Embodiment 162: The kit of embodiment 161, wherein the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of CD27, CD70, CD40, CD40LG, inducible T cell costimulator (ICOS, CD278), inducible T cell costimulator 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), and poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155).

[0172] Embodiment 163: A kit according to any one of embodiments 157 to 162, comprising one or more blockers, antagonists, or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors.

[0173] Embodiment 164: Kit embodiment 163, wherein the NK cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of killer-cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3), killer-cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer-cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), and killer-cell lectin-like receptor D1 (KLRD1, CD94).

[0174] Embodiment 165: A kit according to any one of embodiments 157 to 164, comprising one or more agonists or activators of one or more NK cell-stimulatory immune checkpoint proteins or receptors.

[0175] Embodiment 166: The kit described in embodiment 165, wherein the NK cell-stimulating immune checkpoint protein or receptor is selected from CD16, CD226 (DNAM-1), killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), and SLAM family member 7 (SLAMF7).

[0176] Embodiment 167: The kit according to any one of embodiments 157 to 166, wherein the one or more immune checkpoint inhibitors comprise a proteinaceous inhibitor of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4.

[0177] Embodiment 168: The protein inhibitor of CTLA4 is 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, ATO 168. The kit of embodiment 167, wherein the antibody is selected from the group consisting of R-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), and AK-104 (CTLA4 / PD-1).

[0178] Embodiment 169: The proteinaceous inhibitor of PD-L1 (CD274) or PD-1 (PDCD1) is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, AB122 (zimbarelimab), 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, HL X-10, MGA-012, BI-754091, AGEN-2034 (balstilimab), JS-001 (toripalimab), JNJ-63723283, genolimuzumab (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 (dostallimab), 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-245 0, 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), 168. The kit of embodiment 167, wherein the antibody is selected from the group consisting of 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 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), and INBRX-105 (4-1BB / PDL1).

[0179] Embodiment 170: A kit described in any one of embodiments 157 to 169, wherein the one or more immune checkpoint inhibitors comprise a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4.

[0180] Embodiment 171: The kit described in embodiment 170, wherein the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181.

[0181] Embodiment 172: The kit of embodiment 170, wherein the small molecule inhibitor of CTLA4 comprises BPI-002.

[0182] Embodiment 173: The kit of any one of embodiments 146 to 172, further comprising one or more antiviral agents.

[0183] Embodiment 174: The kit of embodiment 173, wherein the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors. Methods for treating or preventing HIV

[0184] Embodiment 175: A method for inducing an immune response against human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering a pharmaceutical composition according to any one of embodiments 124 to 130 or an immunogenic composition according to any one of embodiments 117 to 121.

[0185] Embodiment 176: A method for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described in any one of embodiments 124 to 130, or an immunogenic composition described in any one of embodiments 117 to 121.

[0186] Embodiment 177: The method of embodiment 175 or 176, comprising administering a single fusion polypeptide, or a polynucleotide or viral expression vector encoding the fusion polypeptide, wherein the fusion polypeptide comprises two or more multivalent polypeptide segments, e.g., bivalent polypeptide segments.

[0187] Embodiment 178: The method of embodiment 175 or 176, wherein two or more fusion polypeptides, or two or more viral expression vectors encoding fusion polypeptides, are administered simultaneously or in parallel to the subject.

[0188] Embodiment 179: The method of any one of embodiments 175 to 178, wherein the two or more fusion polypeptides, or the two or more polynucleotides, or the two or more viral expression vectors encoding the fusion polypeptides, are in the form of a bivalent antigen composition.

[0189] Embodiment 180: To a subject, 1) fusion polypeptides linked or connected in N-terminal to C-terminal order, optionally by one or more linkers; SEQ ID NOs: 70, 76, 94, 151 and 161, or one or more fusion polypeptides, or polynucleotides encoding fusion polypeptides, or viral expression vectors expressing fusion polypeptides, comprising or consisting of the polypeptide segments of SEQ ID NOs: 71, 77, 95, 152, and 162; 2) fusion polypeptides linked or connected in N-terminal to C-terminal order, optionally by one or more linkers; SEQ ID NOs: 188, 305, 28, 41, 294, 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 188, 305, 28, 41 and 294, SEQ ID NOs: 4, 176, 11, 319, 259, 282, 223, 213 and 37, SEQ ID NOs: 189, 306, 29, 42, 295, 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 189, 306, 29, 42 and 295, SEQ ID NOs: 5, 177, 12, 320, 260, 283, 224, 214 and 38, SEQ ID NOs: 305, 319, 259, 282, 223, 213, 294, 176 and 188, SEQ ID NOs: 306, 320, 260, 283, 224, 214, 295, 177 and 189, SEQ ID NOs: 305, 294, 223, 213, 176, 259, 319, 188 and 282, SEQ ID NOs: 306, 295, 224, 214, 177, 260, 320, 189 and 283, SEQ ID NOs: 305, 294, 319, 259, 282, 223, 176 and 188, SEQ ID NOs: 306, 295, 320, 260, 283, 224, 177 and 189, SEQ ID NOs: 305, 223, 294, 176, 259, 319, 188 and 282, or one or more fusion polypeptides, or polynucleotides encoding fusion polypeptides, or viral expression vectors expressing fusion polypeptides, comprising or consisting of the polypeptide segments of SEQ ID NOs: 306, 224, 295, 177, 260, 320, 189, and 283.

[0190] Embodiment 181: To a subject, 1) one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430; 2) the method according to any one of embodiments 175 to 180, comprising administering one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410.

[0191] Embodiment 182: A method comprising administering to a subject one or more fusion polypeptides, or polynucleotides encoding fusion polypeptides, or viral expression vectors expressing fusion polypeptides, wherein the fusion polypeptides are linked or connected in N-terminal to C-terminal order, optionally by one or more linkers. Sequence numbers 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 NOs: 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 The method according to any one of embodiments 175 to 178, comprising or consisting of the polypeptide segments of SEQ ID NOs: 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.

[0192] Embodiment 183: The method of any one of embodiments 175 to 182, comprising administering to a subject one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, 430 to 435, 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 to 377, 407 to 411, 422 to 424, 430 to 435.

[0193] Embodiment 184: The method of any one of embodiments 175 to 183, comprising administering to the subject one or more adenoviral vectors, wherein each adenoviral vector comprises one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, 430 to 435, 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 to 377, 407 to 411, 422 to 424, 430 to 435.

[0194] Embodiment 185: Comprises administering to a subject one or more viral vectors, wherein each viral vector is selected from the group consisting of SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 361 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or 184. The method of any one of embodiments 175 to 183, comprising 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 NOs: 409 and 410.

[0195] Embodiment 186: The method of any one of embodiments 175 to 185, wherein the subject is infected with, suspected of being infected with, or at risk of being infected with HIV-1.

[0196] Embodiment 187: The method of any one of embodiments 175 to 186, wherein the subject is chronically infected with HIV-1.

[0197] Embodiment 188: The method of any one of embodiments 175 to 187, wherein the subject is acutely infected with HIV-1.

[0198] Embodiment 189: The method of any one of embodiments 175 to 188, wherein the subject has HIV-1 infection at Fiebig stage IV or earlier, such as Fiebig stage III, Fiebig stage II, or Fiebig stage I.

[0199] Embodiment 190: The method of any one of embodiments 175 to 189, wherein the composition is administered via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., oral, nasal, rectal, vaginal).

[0200] Embodiment 191: About 10 per administration 3 ~about 10 15 Viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (vp), for example, about 10 4 ~about 10 7 Viral FFU or PFU or IU or vp, for example, about 10 3 ~about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 191. The method of any one of embodiments 175-190, comprising administering viral FFU or PFU or IU or vp.

[0201] Embodiment 192: A prime-boost regimen comprising: (i) 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.); or (ii) The method of any one of embodiments 175-191, comprising a prime-boost regimen comprising one or more repeats (e.g., prime-boost-prime-boost, etc.) of administering a priming composition at a first time point and a boosting composition at a second time point.

[0202] Embodiment 193: The method of embodiment 192, wherein the administration of the priming composition and the one or more boosting compositions is spaced apart by at least 1 week, 2 weeks, 3 weeks, or 1 month, for example, by at least 2, 3, 4, 5, or 6 months.

[0203] Embodiment 194: The method of embodiment 192 or 193, wherein the priming composition and the boosting composition comprise the same immunogenic composition.

[0204] Embodiment 195: The method of embodiment 192 or 193, wherein the priming composition and the boosting composition comprise different immunogenic compositions.

[0205] Embodiment 196: The method of embodiment 192 or 193, wherein the priming composition and the boosting composition comprise the same one or more fusion polypeptides and the same polynucleotide or viral expression vector.

[0206] Embodiment 197: The method of embodiment 192 or 193, wherein the priming composition and the boosting composition comprise different fusion polypeptides and / or different polynucleotides or viral expression vectors.

[0207] Embodiment 198: The method of embodiment 197, comprising priming with a first polynucleotide or viral expression vector and boosting with a second polynucleotide or viral expression vector.

[0208] Embodiment 199: The prime-boost regimen comprises: a) priming with a viral expression vector and boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA; b) priming with a polynucleotide that is DNA, cDNA, mRNA, or self-replicating RNA and boosting with a viral expression vector; c) 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 family; d) 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 family; e) priming with a first attenuated defective viral expression vector and boosting with a second replicating attenuated viral expression vector, wherein the first and second replicating attenuated viral expression vectors are from the same, related, or unrelated taxonomic family; f) priming with a replication-deficient viral expression vector and boosting with a replication-attenuated viral expression vector; g) priming with a replication-attenuated viral expression vector and boosting with a replication-deficient viral expression vector; h) priming with a viral expression vector for lymphocytic choriomeningitis mammarenavirus (LCMV) and boosting with a viral expression vector for Pichinde mammarenavirus; i) priming with a viral expression vector of Pichinde Mammarenavirus and boosting with a viral expression vector of Lymphocytic Choriomeningitis Mammarenavirus (LCMV); j) priming with an arenavirus viral expression vector and boosting with an adenovirus viral expression vector; or k) The method of any one of embodiments 192 to 198, comprising priming with an adenovirus viral expression vector and boosting with an arenavirus viral expression vector.

[0209] Embodiment 200: The method of any one of embodiments 175 to 199, wherein the subject is not receiving antiretroviral therapy (ART) or ART is discontinued prior to administration of the one or more compositions.

[0210] Embodiment 201: The method of any one of embodiments 175 to 200, wherein ART is discontinued after one or more administrations of the composition.

[0211] Embodiment 202: The method of any one of embodiments 175 to 201, further comprising administering to the subject one or more additional therapeutic agents, for example, two, three, four, or more additional therapeutic agents.

[0212] Embodiment 203: The method of embodiment 202, comprising co-administering one or more agents that activate latent HIV, such as one or more latent reactivating agents (LRAs).

[0213] Embodiment 204: The method of embodiment 202 or 203, wherein the one or more LRAs are selected from the group consisting of agonists or activators of one or more Toll-like receptors (TLRs), histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitors of apoptosis proteins (IAP) antagonists, and a second mitochondria-derived activator of caspases (SMAC) mimic.

[0214] Embodiment 205: The method of any one of embodiments 202 to 204, comprising co-administering one or more agonists or activators of one or more Toll-like receptors (TLRs).

[0215] Embodiment 206: The method of embodiment 205, wherein the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0216] Embodiment 207: The method of embodiment 205 or 206, wherein the TLR7 agonist is selected from the group consisting of GS9620 (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).

[0217] Embodiment 208: The method of any one of embodiments 202 to 207, comprising co-administering one or more interleukin receptor agonists of interleukins selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, GM-CSF, and FLT3LG.

[0218] Embodiment 209: The method of embodiment 208, comprising co-administering one or more cytokines selected from the group consisting of 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.

[0219] Embodiment 210: The method of any one of embodiments 202 to 209, comprising co-administering one or more innate immune activators.

[0220] Embodiment 211: The method of embodiment 210, wherein the one or more innate immune activators comprise non-coding immunostimulatory polynucleotides (e.g., pathogen-activating molecular patterns (PAMPs), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides, and immunostimulatory RNA (isRNA, e.g., CV8102)), agonists of receptors selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain-containing 2 (NOD2).

[0221] Embodiment 212: The method of any one of embodiments 202 to 211, comprising co-administering one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors, and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors.

[0222] Embodiment 213: The one or more immune checkpoint proteins or receptors are selected from the group consisting of CD27, CD70, CD40, CD40LG, CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4), CD276 (B7H3), V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), immunoglobulin superfamily member 11 (IGSF11, VSIG3), natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6), and HERV-H. Long-Term Receptor Associated 2 (HHLA2, B7H7), Inducible T Cell Costimulator (ICOS, CD278), Inducible T Cell Costimulator Ligand (ICOSLG, B7H2), TNF Receptor Superfamily Member 4 (TNFRSF4, OX40), TNF Superfamily Member 4 (TNFSF4, OX40L), TNFRSF8 (CD30), TNFSF8 (CD30L), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL), TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML), and CD272 (B and T Lymphocyte-Associated (BTLA) ), TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF), TNFRSF18 (GITR), TNFSF18 (GITRL), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), CD274 (CD274, PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), CD80 (B7-1), CD28, nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155), PVR-related immunoglobulin domain-containing (PVRIG, CD112R),T cell immunoreceptor containing Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), galectin 9 (LGALS9), lymphocyte activation 3 (LAG3, CD223), signaling lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150), lymphocyte antigen 9 (LY9, CD229, SLAMF3), and SLAM family member 6 (SLAM F6, CD352), SLAM family member 7 (SLAMF7, CD319), UL16 binding protein 1 (ULBP1), UL16 binding protein 2 (ULBP2), UL16 binding protein 3 (ULBP3), retinoic acid early transcript 1E (RAET1E; ULBP4), retinoic acid early transcript 1G (RAET1G; ULBP5), retinoic acid early transcript 1L (RAET1L; ULBP6), lymphocyte antigen 3 (CD223), killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1), killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C), killer cell lectin-like receptor C3 (KLRC3, NKG2E), killer cell lectin-like receptor C4 (KLRC4, NKG2F), killer cell immunoglobulin-like receptor, two Ig domains, and a long cytoplasmic tail 213. The method of embodiment 212, wherein the target polypeptide is selected from the group consisting of: killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1), killer cell lectin-like receptor D1 (KLRD1), and SLAM family member 7 (SLAMF7).

[0223] Embodiment 214: The method of embodiment 212 or 213, comprising co-administering one or more blockers, antagonists, or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors.

[0224] Embodiment 215: The T cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of CD274 (CD274, PDL1, PD-L1), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), programmed cell death 1 (PDCD1, PD1, PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), CD276 (B7H3), and V-set domain-containing T cell inhibitory receptor (VCR). 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), and Ig and Ig receptors. 215. The method of embodiment 214, wherein the T cell immunoreceptor is selected from the group consisting of a T cell immunoreceptor comprising a TIM domain (TIGIT), lymphocyte activation 3 (LAG3, CD223), hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3), galectin 9 (LGALS9), killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), and killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1).

[0225] Embodiment 216: The method of embodiment 212 or 213, comprising co-administering one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors.

[0226] Embodiment 217: The method of embodiment 216, wherein the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of CD27, CD70, CD40, CD40LG, inducible T cell costimulator (ICOS, CD278), inducible T cell costimulator 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), and poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155).

[0227] Embodiment 218: The method of embodiment 212 or 213, comprising co-administering one or more blockers, antagonists, or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors.

[0228] Embodiment 219: The method of embodiment 218, wherein the NK cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A), and killer cell lectin-like receptor D1 (KLRD1, CD94).

[0229] Embodiment 220: The method of embodiment 212 or 213, comprising co-administering one or more agonists or activators of one or more NK cell-stimulatory immune checkpoint proteins or receptors.

[0230] Embodiment 221: The method of embodiment 220, wherein the NK cell-stimulating immune checkpoint protein or receptor is selected from CD16, CD226 (DNAM-1), killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314), and SLAM family member 7 (SLAMF7).

[0231] Embodiment 222: The method of any one of embodiments 212 to 215, wherein the one or more immune checkpoint inhibitors comprise a proteinaceous inhibitor of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4.

[0232] Embodiment 223: The protein inhibitor of CTLA4 is 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, AT 223. The method of embodiment 222, wherein the antibody is selected from the group consisting of OR-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), and AK-104 (CTLA4 / PD-1).

[0233] Embodiment 224: The proteinaceous inhibitor of PD-L1 (CD274) or PD-1 (PDCD1) is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, AB122 (zimbarelimab), 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, H LX-10, MGA-012, BI-754091, AGEN-2034 (balstilimab), JS-001 (toripalimab), JNJ-63723283, genolimuzumab (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 (dostallimab), MSB-2311, JTX-4014, BG B-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-24 50, 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 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), and INBRX-105 (4-1BB / PDL1).

[0234] Embodiment 225: The method of any one of embodiments 212 to 215, wherein the one or more immune checkpoint inhibitors comprise a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4.

[0235] Embodiment 226: The method of embodiment 225, wherein the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181.

[0236] Embodiment 227: The method of embodiment 225, wherein the small molecule inhibitor of CTLA4 comprises BPI-002.

[0237] Embodiment 228: The method of any one of embodiments 202 to 227, further comprising administering to the subject one or more antiviral agents.

[0238] Embodiment 229: The method of embodiment 228, wherein the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors.

[0239] Embodiment 230: The method of any one of embodiments 202 to 229, further comprising administering to the subject one or more anti-HIV antibodies or antigen-binding fragments thereof.

[0240] Embodiment 231: The method of embodiment 230, wherein the one or more anti-HIV antibodies or antigen-binding fragments thereof bind to HIV gp120.

[0241] Embodiment 232: The method of embodiment 230 or 231, wherein the anti-HIV antibody or antigen-binding fragment thereof comprises a broadly neutralizing antibody.

[0242] Embodiment 233: The method of any one of embodiments 230 to 232, wherein one or more anti-HIV antibodies or antigen-binding fragments thereof that bind, inhibit, and / or neutralize HIV compete with or comprise the VH and VL variable domains of a broadly neutralizing antibody (bNAb) against HIV.

[0243] Embodiment 234: The one or more anti-HIV antibodies or antigen-binding fragments thereof that bind, inhibit, and / or neutralize HIV are: i. a third variable loop (V3) and / or a high mannose patch comprising an N332 oligomannose glycan; ii.CD4 binding site (CD4bs), iii. the second variable loop (V2) and / or the Env trimer vertex; iv. the gp120 / gp41 interface, or v. The method of any one of embodiments 230-233, which binds to an epitope or region of gp120 selected from the group consisting of silent faces of gp120.

[0244] Embodiment 235: An antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV, binds to an epitope or region of gp120 within the third variable loop (V3) and / or the high mannose patch comprising the N332 oligomannose glycan, and is selected from the group consisting of GS-9722, PGT-121, PGT-122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-128, PGT-130, PGT-133, PGT-134, PGT-135, PGT-136, PGT-137, PGT-138, 235. The method of any one of embodiments 230-234, which competes with or comprises VH and VL regions from an antibody selected from the group consisting of PGT-139, 10-1074, VRC24, 2G12, BG18, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, and VRC29.03.

[0245] Embodiment 236: The antibody or antigen-binding fragment thereof binds to an epitope or region of gp120 in the CD4 binding site (CD4bs) and is selected from the group consisting of b12, F105, VRC01, VRC07, VRC07-523, VRC03, VRC06, VRC06b01, VRC08, VRC0801, NIH45-46, GS-9723, 3BNC117, 3BNC60, VRC-PG04, PGV0 236. The method of any one of embodiments 230 to 235, wherein the antibody competes with or comprises a VH and VL region from an antibody selected from the group consisting of: N6, N49-P7, NC-Cowl, IOMA, CH235 and CH235.12, N49P6, N49P7, N49P11, N49P9, and N60P25.

[0246] Embodiment 237: The method of any one of embodiments 230 to 236, wherein the antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV binds to an epitope or region of gp120 in the second variable loop (V2) and / or Env trimer apex and competes with or comprises VH and VL regions from an antibody selected from the group consisting of PG9, PG16, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGT-145, CH01, CH59, PGDM1400, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.25, PCT64-24E, and VRC38.01.

[0247] Embodiment 238: The method of any one of embodiments 230 to 237, wherein the antibody or antigen-binding fragment binds to an epitope or region of gp120 at the gp120 / gp41 interface and competes with or comprises VH and VL regions from an antibody selected from the group consisting of PGT-151, CAP248-2B, 35O22, 8ANC195, ACS202, VRC34, and VRC34.01.

[0248] Embodiment 239: The method of any one of embodiments 230 to 238, wherein the antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV binds to an epitope or region on the gp120 silent face and competes with or comprises a VH and VL region from an antibody selected from the group consisting of VRC-PG05 and SF12.

[0249] Embodiment 240: The method of any one of embodiments 230 to 239, wherein the antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV binds to an epitope or region of gp41 within the membrane proximal region (MPER).

[0250] Embodiment 241: The method of any one of embodiments 230 to 240, wherein the antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV binds to an epitope or region of gp41 within the membrane proximal region (MPER) and competes with or comprises VH and VL regions from an antibody selected from the group consisting of 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, and LN01.

[0251] Embodiment 242: The method of any one of embodiments 230 to 241, wherein the antibody or antigen-binding fragment thereof that binds, inhibits, and / or neutralizes HIV binds to an epitope or region of a gp41 fusion peptide and competes with or comprises a VH and VL region from an antibody selected from the group consisting of VRC34 and ACS202.

[0252] Embodiment 243: The method of any one of embodiments 175 to 242, wherein after one or more administrations of one or more of the compositions, optionally in combination with one or more additional therapeutic agents, the subject does not exhibit symptoms of HIV or AIDS for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or more, in the absence of antiretroviral therapy (ART).

[0253] Embodiment 244: The method of any one of embodiments 175 to 243, wherein after one or more administrations of one or more of the compositions, optionally in combination with one or more additional therapeutic agents, the subject has a viral load of less than 500, e.g., less than 400, less than 300, less than 200, less than 100, less than 50 copies / ml of blood in the absence of antiretroviral therapy (ART) for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or more. Methods for Immunogen Design

[0254] Embodiment 245: A method for designing a fusion polypeptide capable of inducing an immune response against one or more viral target antigens, comprising: a) identifying in silico one or more regions of sequence conservation in a population of polypeptide sequences encoded by viral genes, the population being derived from a patient-to-patient viral population; b) identifying in silico the two most common polypeptide sequences from the one or more conserved regions identified in step a) and generating multivalent polypeptide segments from the conserved regions.

[0255] Embodiment 246: The method of embodiment 245, wherein the multivalent polypeptide segment is a bivalent polypeptide segment.

[0256] Embodiment 247: The method of embodiment 245 or 246, further comprising step c) arranging the polypeptide segments in one or more contiguous fusion polypeptides such that the junctions connecting the polypeptide segments reduce or avoid the creation of epitopes capable of binding to human MHC class I or human MHC class II molecules, for example with a predicted binding affinity IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in the population of polypeptide segments.

[0257] Embodiment 248: The method of any one of embodiments 245 to 247, further comprising inserting a linker between polypeptide segment junctions predicted to create epitopes capable of binding to human MHC class I or human MHC class II molecules.

[0258] Embodiment 249: After step b) and before step c), d) in silico identifying polypeptide segments within one or more regions of sequence conservation identified in step a) that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% of the population of polypeptide segments; e) generating polypeptide segments identified in step a) that contain one or more sequence conserved regions that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in the population of polypeptide segments.

[0259] Embodiment 250: The method of any one of embodiments 245 to 249, further comprising, after step b) and before step c), a step of reducing or removing viral polypeptide 9-mers having at least 55% (5 of 9 amino acid residues), such as at least 65% (6 of 9 amino acid residues), for example at least 75% (7 of 9 amino acid residues), for example at least 85% (8 of 9 amino acid residues) amino acid sequence identity to a human protein.

[0260] Embodiment 251: The method of any one of embodiments 245 to 250, further comprising, after step b) and before step c), providing one or more polypeptide segments that are known or predicted to bind to human MHC class II molecules, for example, with a predicted binding affinity IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in a population of polypeptide segments.

[0261] Embodiment 252: The method of any one of embodiments 245 to 251, further comprising, after step b) and before step c), identifying sequence variance in a second population of polypeptide sequences encoded by viral genes within one or more regions of sequence conservation identified in step a), wherein the second population is derived from an intra-patient viral population.

[0262] Embodiment 253: The method of embodiment 252, wherein the sequence variance from the intra-patient viral population is determined by deep sequencing or next generation sequencing.

[0263] Embodiment 254: A method for designing a fusion polypeptide capable of inducing an immune response against one or more viral target antigens, comprising: a) identifying in silico one or more regions of sequence conservation in a first population of polypeptide sequences encoded by viral genes, wherein the first population is derived from a patient-to-patient viral population; b) identifying in silico the two most common polypeptide sequences from one or more of the conserved regions identified in step a); c) in silico identifying polypeptide segments within one or more regions of sequence conservation identified in step a) that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% of the population of polypeptide segments; d) generating polypeptide segments identified in step a) that contain one or more sequence conserved regions that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% of the population of polypeptide segments; e) removing the viral polypeptide 9-mer segments produced in step d) that are determined to have at least 55% (5 out of 9 amino acid residues), such as at least 65% (6 out of 9 amino acid residues), such as at least 75% (7 out of 9 amino acid residues), such as at least 85% (8 out of 9 amino acid residues) amino acid sequence identity to a human protein to obtain retained viral polypeptide segments; f) arranging the retained polypeptide segments into one or more contiguous fusion polypeptides such that the junctions connecting the polypeptide segments avoid or reduce the creation of epitopes capable of binding to human MHC class I or human MHC class II molecules, e.g., with a predicted binding affinity IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in the population of polypeptide segments.

[0264] Embodiment 255: A method for designing a fusion polypeptide capable of inducing an immune response against one or more viral target antigens, comprising: a) identifying in silico one or more regions of sequence conservation in a first population of polypeptide sequences encoded by viral genes, wherein the first population is derived from a patient-to-patient viral population; b) optionally identifying in silico the two most common polypeptide sequences from one or more conserved regions identified in step a); c) identifying sequence variance in a second population of polypeptide sequences encoded by viral genes within one or more regions of sequence conservation identified in step a), wherein the second population is derived from an intra-patient viral population; d) in silico identifying polypeptide segments within one or more regions of sequence conservation identified in step a) that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% of the population of polypeptide segments; e) generating polypeptide segments identified in step a) that contain one or more sequence conserved regions that are predicted to bind to human MHC class I molecules with an IC50 value of less than about 1000 nM or with a percentile rank within the top 5% of the population of polypeptide segments; f) removing the viral polypeptide 9-mer segments produced in step e) that are determined to have at least 55% (5 out of 9 amino acid residues), such as at least 65% (6 out of 9 amino acid residues), such as at least 75% (7 out of 9 amino acid residues), for example at least 85% (8 out of 9 amino acid residues) amino acid sequence identity to a human protein to obtain retained viral polypeptide segments; g) arranging the retained polypeptide segments into one or more contiguous fusion polypeptides such that the junctions connecting the polypeptide segments avoid or reduce the creation of epitopes capable of binding to human MHC class I or human MHC class II molecules, e.g., with a predicted binding affinity IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in the population of polypeptide segments.

[0265] Embodiment 256: The method of embodiment 255, wherein the sequence variance from the intra-patient viral population is determined by deep sequencing or next generation sequencing.

[0266] Embodiment 257: The method of any one of embodiments 254 to 256, further comprising providing one or more polypeptide segments that are known or predicted to bind to a human MHC class II molecule, for example, with a predicted binding affinity IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in a population of polypeptide segments.

[0267] Embodiment 258: The method of any one of embodiments 254 to 257, further comprising inserting a linker between polypeptide segment junctions predicted to create epitopes capable of binding to human MHC class I or human MHC class II molecules.

[0268] Embodiment 259: A method for producing a multivalent antigen, the method comprising: (a) aligning a collection of viral proteome sequences; (b) for each sequence in the alignment, creating a set of 9-amino acid subsequences ("9-mers") starting from the N-terminal amino acid, where each subsequence overlaps the preceding subsequence by 8 amino acids, such that each sequence of length l in the alignment contains (l-8) 9-mers; (c) calculating the frequency of each unique 9-mer starting at position i in each sequence of the alignment and identifying two or more of the most common 9-mers at each position, where (c)(1) the frequency is calculated as the number of unique 9-mers at position i in the alignment divided by the total number of sequences in the alignment; (d) calculating the multivalent conservation at each position by summing the fraction of sequences in the alignment that contain any of two or more of the most common 9-mers; (e) creating an alignment of conserved regions by extracting sequences in the alignment that have greater than 80% or greater than 90% multivalent conservation; (f) determining the frequency of each unique 9-mer pair at each position in the alignment of conserved regions; (g) connecting 9-mer pairs at adjacent positions in the alignment of conserved regions that share an overlap of 8 amino acids; (h) creating a directed acyclic graph in which each 9mer pair is a node, edges between adjacent nodes are formed from connected 9mer pairs at adjacent locations, and the weight of each edge is equal to the frequency of the downstream 9mer pair; Adding a source node and connecting it with all nodes in the first position; Add a sink node and connect it with all nodes in the last position; Disable all weights and (i) finding an optimal path in a directed acyclic graph from a source node to a sink node, where the optimal path is defined in terms of the sum of the frequencies of all 9-mer pairs in the directed acyclic graph; (j) constructing a multivalent antigen by connecting two or more 9-mers that are in adjacent positions within an optimal multivalent 9-mer pathway where they share an overlap of eight amino acids, thereby creating two or more sequences of connected 9-mers that together form the multivalent antigen; (k) optionally, rearranging the polypeptide segments to reduce or avoid the creation of harmful epitopes at junctions between the polypeptide segments, thereby constructing in silico a set of multivalent amino acid sequences within structurally conserved regions of a population of viral proteome sequences.

[0269] Embodiment 260: The method of embodiment 259, wherein the multivalent reservoir is a bivalent reservoir and the multivalent antigen is a bivalent antigen.

[0270] Embodiment 261: In step (a), the conserved region is (i) removing segments less than 35 amino acids in length, e.g., from 9 amino acids to 10, 15, 20, 25, 30, or 35 amino acids in length; (ii) removing segments determined to have less than 90% multivalent (e.g., bivalent) conservation; (iii) removing segments that are determined to be weakly immunogenic or non-immunogenic, e.g., as demonstrated in vitro or in vivo; and / or (iv) the inclusion of an additional segment determined to be immunogenic, e.g., as demonstrated in vitro or in vivo.

[0271] Embodiment 262: The method described in any one of embodiments 259 to 261, wherein the step of rearranging peptide segments to reduce or avoid the creation of harmful epitopes is carried out by a method comprising one or more of in silico HLA binding analysis and human proteome cross-recognition analysis.

[0272] Embodiment 263: The method of any one of embodiments 259 to 262, further comprising inserting a linker sequence between one or more adjacent segments.

[0273] Embodiment 264: The method of any one of embodiments 259 to 263, wherein the method further comprises improving the multivalent (e.g., bivalent) antigen produced in step (h) by removing junction 9-mers that bind to specific HLA alleles with a predicted IC50 value of less than about 1000 nM or with a percentile rank within the top 5% in the population of polypeptide segments.

[0274] Embodiment 265: The method of any one of embodiments 259 to 264, wherein the method further comprises improving the multivalent (e.g., bivalent) antigen produced in step (h) by removing 9-mers that have at least 55% (5 out of 9 amino acid residues), such as at least 65% (6 out of 9 amino acid residues), such as at least 75% (7 out of 9 amino acid residues), for example at least 85% (8 out of 9 amino acid residues) amino acid sequence identity with a human peptide, or that have the same T cell receptor (TCR)-facing residues as the human protein.

[0275] Embodiment 266: The method further comprises improving the multivalent (e.g., bivalent) antigen produced in step (h) to generate sufficient T cell epitopes to cover intra-patient viral diversity, the method comprising: a) identifying viral quasispecies variants in a biological sample obtained from a subject; and b) (i) at each 9-mer position in a multivalent (e.g., bivalent) antigen, determining a corresponding 9-mer subsequence from multiple sequencing reads that completely cover the position; (ii) extracting a 9-mer subsequence; (iii) determining intra-patient amino acid variants from the sequence of the multivalent (e.g., bivalent) antigen produced in step (h) by a method comprising: aligning the extracted 9-mer subsequences against the sequence of the multivalent (e.g., bivalent) antigen and determining the presence of any mismatches.

[0276] Embodiment 267: The method described in embodiment 266, wherein the viral quasispecies is identified by a method comprising sequencing viral DNA, assembling multiple sequence reads to create a target consensus sequence, aligning each read in the multiple reads to the target consensus sequence, and mapping the aligned reads of interest to a reference sequence to obtain sequence coordinates.

[0277] Embodiment 268: The method of embodiment 266 or 267, wherein the biological sample is selected from blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, semen, or lymph nodes.

[0278] Embodiment 269: The method of any one of embodiments 266 to 268, wherein the subject is acutely infected with HIV-1.

[0279] Embodiment 270: The method of any one of embodiments 266 to 269, wherein the subject has HIV-1 infection at Fiebig stage IV or earlier, such as Fiebig stage III, Fiebig stage II, or Fiebig stage I.

[0280] Embodiment 271: The method of any one of embodiments 266 to 268, wherein the subject is chronically infected with HIV-1.

[0281] Embodiment 272: The method of any one of embodiments 266 to 271, wherein the subject is receiving antiretroviral therapy (ART).

[0282] Embodiment 273: The method of any one of embodiments 266 to 271, wherein the subject is not receiving antiretroviral therapy (ART).

[0283] Embodiment 274: The method of any one of embodiments 266 to 273, further comprising excluding sequences with pre-existing escape variants.

[0284] Embodiment 275: The method of any one of embodiments 259 to 274, further comprising rearranging the polypeptide segments to reduce or avoid the creation of harmful epitopes at the junctions between the polypeptide segments.

[0285] Embodiment 276: The method described in embodiment 275, wherein the step of rearranging peptide segments to reduce or avoid the creation of harmful epitopes is carried out by a method comprising one or more of in silico HLA binding analysis and human proteome cross-recognition analysis.

[0286] Embodiment 277: The method of any one of embodiments 245 to 276, wherein the one or more viral target antigens are derived from a mammalian virus, for example, a human virus.

[0287] Embodiment 278: The method of any one of embodiments 245 to 277, wherein the one or more viral target antigens are derived from a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papillomavirus (HPV), herpes simplex virus (HSV), Ebola virus, Zika virus, and Chikungunya virus.

[0288] Embodiment 279: The method of any one of embodiments 245 to 278, wherein the patient viral population is derived from a population of patients not receiving antiretroviral therapy (ART).

[0289] Embodiment 280: The method of any one of embodiments 245 to 278, wherein the patient viral population is derived from a population of patients receiving antiretroviral therapy (ART).

[0290] Embodiment 281: The method of any one of embodiments 252 to 280, wherein the intra-patient viral population is derived from a patient not receiving antiretroviral therapy (ART).

[0291] Embodiment 282: The method of any one of embodiments 252 to 280, wherein the intra-patient viral population is derived from a patient receiving antiretroviral therapy (ART).

[0292] Embodiment 283: A fusion polypeptide produced according to the method of any one of embodiments 245 to 282, wherein the fusion polypeptide induces an immune response against a virus in a mammal, such as a human. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A fusion polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 352, 357 to 362, 367, 373, 407 to 411, or 422 to 424, or a sequence that is at least 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 to 352, 357 to 362, 367, 373, 407 to 411, 422 to 424, and 431 to 435. (Item 2) 2. The fusion polypeptide of item 1, wherein the fusion protein does not contain polypeptide segments encoded by the HIV-1 Tat, Rev, Vif, Vpr and / or Vpu genes. (Item 3) the fusion protein (i) an HIV-1 Gag amino acid sequence or a partial sequence thereof corresponding to an amino acid residue position selected from 1 to 30, 54 to 127, 138 to 146, 370 to 428, and 445 to 500, wherein the amino acid positions are based on SEQ ID NO: 404; (ii) an HIV-1 Nef amino acid sequence or a subsequence thereof corresponding to amino acid residue positions selected from 1 to 63, 103 to 116, and 155 to 206, wherein the amino acid positions are based on SEQ ID NO: 405; (iii) HIV-1 corresponding to an amino acid residue position selected from 1 to 27, 53 to 58, 84 to 112, 138 to 234, 269 to 474, 490 to 501, and 611 to 856 an HIV-1 Env amino acid sequence or a subsequence thereof, wherein the amino acid positions are based on SEQ ID NO: 403; and / or (iv) The fusion polypeptide of item 1 or 2, wherein the fusion polypeptide does not contain one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 Pol amino acid sequence corresponding to an amino acid residue position selected from 1 to 55, 118 to 128, 321 to 325, 355 to 366, 432 to 541, 607 to 641, 667 to 682, 709 to 746, 828 to 833, or 921 to 930, based on SEQ ID NO: 406, and wherein the fusion polypeptide does not contain one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 Pol amino acid sequence corresponding to the amino acid residue positions selected from 1 to 55, 118 to 128, 321 to 325, 355 to 366, 432 to 541, 607 to 641, 667 to 682, 709 to 746, 828 to 833, or 921 to 930, based on SEQ ID NO: 406. (Item 4) 4. The fusion polypeptide according to any one of items 1 to 3, wherein the fusion polypeptide does not comprise one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 amino acid sequence of SEQ ID NOs: 437 to 461, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NOs: 437 to 461, or a subsequence thereof. (Item 5) 5. The fusion polypeptide of any one of items 1 to 4, wherein the fusion polypeptide does not contain an HIV-1 Pol polypeptide segment comprising the amino acid sequence YMDD (SEQ ID NO: 462) or YVDD (SEQ ID NO: 463). (Item 6) 6. The fusion polypeptide of item 5, wherein the fusion polypeptide does not contain one or more amino acid sequences selected from SEQ ID NOs: 215, 216, 217, 218, 219, and 220. (Item 7) 7. The fusion polypeptide according to item 5 or 6, wherein the fusion polypeptide does not comprise one or more amino acid sequences selected from SEQ ID NOs: 209, 210, 211, 212, 213, 214, 343 and 344. (Item 8) 8. The fusion polypeptide according to any one of items 1 to 7, comprising an N-terminal signal peptide or leader sequence. (Item 9) 9. The fusion polypeptide of item 8, wherein the signal peptide or leader sequence is derived from a source protein selected from the group consisting of colony-stimulating factor 2 (CSF2, GM-CSF), tissue-type plasminogen activator (PLAT, t-PA), CC-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). (Item 10) 10. A polynucleotide encoding one or more fusion polypeptides according to any one of items 1 to 9. (Item 11) 11. The polynucleotide of item 10, wherein the polynucleotide comprises cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). (Item 12) 12. The polynucleotide of claim 11, wherein the polynucleotide comprises a self-replicating or self-amplifying alphavirus replicon. (Item 13) 13. The polynucleotide of any one of items 10 to 12, comprising the nucleic acid sequence of any one of SEQ ID NOs: 414 to 418, or a sequence which 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: 414 to 418. (Item 14) A lipid nanoparticle (LNP) comprising the polynucleotide according to any one of items 10 to 13. (Item 15) 14. An expression cassette comprising the polynucleotide of any one of items 10 to 13 operably linked to one or more regulatory sequences. (Item 16) 16. The expression cassette of item 15, wherein the polynucleotide is operably linked to and under the control of a constitutive promoter. (Item 17) 17. The expression cassette according to item 15 or 16, wherein the promoter is selected from the group consisting of a CMV promoter, a CAG promoter, and an EF1a promoter. (Item 18) A vector comprising one or more polynucleotides according to any one of Items 10 to 13 or the expression cassette according to any one of Items 15 to 17. (Item 19) 19. The vector according to item 18, wherein the vector is a plasmid vector, a bacterial vector, or a viral vector. (Item 20) 20. The vector according to item 18 or 19, wherein the vector is a viral vector. (Item 21) 21. The vector according to any one of items 18 to 20, wherein the viral vector is derived from a DNA virus or an RNA virus. (Item 22) 22. The vector according to any one of Items 18 to 21, wherein the viral 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. (Item 23) 23. The vector of any one of items 18 to 22, wherein the viral 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)), Paramyxoviridae (e.g., measles virus), Flaviviridae (e.g., yellow fever virus), Rhabdoviridae (e.g., vesiculovirus, e.g., Marabavesiculovirus), and Togaviridae (e.g., alphavirus). (Item 24) 24. The vector of any one of items 18 to 23, wherein the viral vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Kali 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). (Item 25) 25. The vector of item 24, wherein the viral vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus). (Item 26) 24. The vector according to any one of items 18 to 23, wherein the viral vector is a human adenovirus or a simian adenovirus (for example, a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus adenovirus). (Item 27) The viral vector is selected from the group consisting of adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (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), ChAd3 8(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)), Go 27. The vector of item 26, which is an adenovirus vector selected from a Lyra adenovirus (e.g., GC44, GC45, GC46) and a Rhesus adenovirus (e.g., RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66). (Item 28) 28. The vector according to any one of items 18 to 27, wherein the viral vector is replication-defective, replication-deficient, replication-attenuated or replication-competent. (Item 29) 29. The vector according to any one of Items 18 to 28, wherein the viral vector is an adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising an amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, and 430 to 435, 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 to 377, 407 to 411, 422 to 424, and 430 to 435. (Item 30) The vector is SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or 30. The vector according to any one of items 18 to 29, comprising 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 sequence of SEQ ID NOs: 409 and 410. (Item 31) 31. The vector of any one of items 18 to 30, further comprising a polynucleotide encoding a cytokine or a functional variant thereof, or a non-coding immunostimulatory polynucleotide. (Item 32) 32. The vector of item 31, comprising 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, GM-CSF, fms-related receptor tyrosine kinase 3 ligand (FLT3LG), and combinations and functional variants thereof. (Item 33) 34. The vector of claim 31, comprising a non-coding immunostimulatory polynucleotide selected from a pathogen-activating molecular pattern (PAMP), a cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide, and an immunostimulatory RNA (isRNA, e.g., CV8102). A host cell comprising one or more polynucleotides according to any one of Items 10 to 13 or one or more vectors according to any one of Items 18 to 33. (Item 35) 35. The host cell of item 34, wherein the one or more polynucleotides are not integrated into the host cell genome, e.g., are episomal. (Item 36) 35. The host cell of item 34, wherein the one or more polynucleotides are integrated into the host cell genome. (Item 37) 37. The host cell according to any one of items 34 to 36, wherein the host cell is a mammalian cell, for example a human cell. (Item 38) 38. The host cell according to any one of items 34 to 37, wherein the host cell is in vitro. (Item 39) The host cell according to any one of Items 34 to 37, wherein the host cell is in vivo. (Item 40) An immunogenic composition comprising one or more of the fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, and a pharmaceutically acceptable carrier. (Item 41) 41. The immunogenic composition according to item 40, comprising two or more of the fusion polypeptides according to any one of items 1 to 9, or two or more polynucleotides according to any one of items 10 to 13, or two or more vectors according to any one of items 18 to 33. (Item 42) 42. The immunogenic composition of item 40 or 41, wherein the one or more polynucleotides are DNA, cDNA, mRNA, or self-replicating RNA. (Item 43) 43. The immunogenic composition according to any one of Items 40 to 42, comprising one or more adenoviral vectors, each adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, and 430 to 435, 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 to 377, 407 to 411, 422 to 424, and 430 to 435. (Item 44) one or more viral vectors, each viral vector comprising: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or 44. The immunogenic composition according to any one of items 40 to 43, comprising one 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 NOs: 409 and 410. (Item 45) 1) one or more fusion polypeptides comprising one or more polynucleotides encoding the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430, or one or more vectors capable of expressing the same; 2) the immunogenic composition according to any one of Items 40 to 43, comprising one or more fusion polypeptides comprising one or more polynucleotides encoding any one of the amino acid sequences of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410, or one or more fusion polypeptides comprising one or more vectors capable of expressing the same. (Item 46) A pharmaceutical composition comprising one or more of the fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, and a pharmaceutically acceptable carrier. (Item 47) 47. The pharmaceutical composition according to item 46, comprising two or more fusion polypeptides, two or more polynucleotides, or two or more vectors. (Item 48) 48. The pharmaceutical composition according to item 46 or 47, further comprising one or more of an adjuvant, an immunostimulant, a detergent, a micelle-forming agent, and an oil. (Item 49) 49. The pharmaceutical composition of item 48, wherein the immunostimulatory agent is selected from a Toll-like receptor (TLR) agonist, a cytokine, a non-coding immunostimulatory polynucleotide, an inhibitor of an inhibitory immune checkpoint protein, or a stimulator of a stimulatory immune checkpoint protein. (Item 50) 50. The pharmaceutical composition of item 49, comprising 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, GM-CSF, FLT3LG, and combinations and functional variants thereof. (Item 51) 50. The pharmaceutical composition of item 49, comprising a non-coding immunostimulatory polynucleotide selected from pathogen-activating molecular patterns (PAMPs), cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides, and immunostimulatory RNA (isRNA, e.g., CV8102). (Item 52) 52. The pharmaceutical composition according to any one of items 46 to 51, formulated for administration via a route selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., oral, nasal, rectal, vaginal). (Item 53) 53. The pharmaceutical composition according to any one of items 46 to 52, formulated as a liquid. (Item 54) 53. The pharmaceutical composition according to any one of items 46 to 52, wherein the composition is lyophilized. (Item 55) A kit comprising one or more unit doses of one or more of the fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, or one or more immunogenic compositions according to any one of items 40 to 45, or one or more pharmaceutical compositions according to any one of items 46 to 54. (Item 56) 56. The kit of item 55, wherein the one or more unit doses are in a single container. (Item 57) 56. The kit of item 55, wherein the one or more unit doses are in two or more separate containers. (Item 58) 58. The kit of any one of items 55 to 57, comprising one or more containers selected from the group consisting of vials, ampoules, and pre-loaded syringes. (Item 59) 59. The kit according to any one of items 55 to 58, comprising one or more containers containing the one or more fusion polypeptides, one or more polynucleotides, or one or more vectors in aqueous solution. (Item 60) 60. The kit according to any one of items 55 to 59, wherein the one or more unit doses are the same. (Item 61) 60. The kit according to any one of items 55 to 59, wherein the one or more unit doses are different. (Item 62) 34. The method of claim 33, wherein the one or more unit doses of the one or more viral vectors are about 10 3 ~about 10 15 Viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (vp), for example, about 10 4 ~about 10 7 Viral FFU or PFU or IU or vp, for example, about 10 3 ~about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 62. The kit according to any one of Items 55 to 61, wherein the viral load is within the range of FFU, PFU, IU, or vp. (Item 63) The kit according to any one of Items 55 to 62, comprising two or more of the fusion polypeptides according to any one of Items 1 to 9, or two or more polynucleotides according to any one of Items 10 to 13, or two or more vectors according to any one of Items 18 to 33. (Item 64) 64. The kit according to any one of Items 55 to 63, comprising one or more polynucleotides encoding the fusion polypeptide or one or more vectors expressing the fusion polypeptide, wherein the fusion polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 345 to 377, 411, 422 to 424, and 430 to 435, 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 to 377, 411, 422 to 424, and 430 to 435. (Item 65) 65. The kit of any one of Items 55 to 64, comprising one or more adenoviral vectors, each adenoviral vector comprising one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, and 430 to 435, 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 to 377, 407 to 411, 422 to 424, and 430 to 435. (Item 66) one or more viral vectors, each viral vector comprising: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or 66. The kit according to any one of items 55 to 65, comprising 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 NOs: 409 and 410. (Item 67) The fusion polypeptide comprises two or more polynucleotides encoding the fusion polypeptide or two or more vectors expressing the fusion polypeptide, 1) an amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430; 2) The kit according to Item 63, comprising an amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410. (Item 68) 67. The kit of any one of items 55 to 66, further comprising one or more unit doses of one or more additional therapeutic agents. (Item 69) 70. The kit of item 68, comprising one or more agents that activate latent HIV, e.g., one or more latent reactivating agents (LRAs). (Item 70) 70. The kit of item 68 or 69, comprising one or more LRAs selected from the group consisting of agonists or activators of one or more Toll-like receptors (TLRs), histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitors of apoptosis proteins (IAP) antagonists, and a second mitochondria-derived activator of caspases (SMAC) mimetic. (Item 71) 71. The kit according to any one of items 68 to 70, comprising one or more agonists or activators of one or more Toll-like receptors (TLRs). (Item 72) 72. The kit of item 71, wherein the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. (Item 73) 73. The kit of item 71 or 72, wherein the TLR7 agonist is selected from the group consisting of GS9620 (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). (Item 74) 74. The kit of any one of items 71 to 73, wherein the TLR9 agonist is selected from the group consisting of AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, ritenimod, 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. (Item 75) 75. The kit of any one of Items 68 to 74, comprising one or more interleukin receptor agonists of interleukins selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, GM-CSF, and FLT3LG. (Item 76) 76. The kit of item 75, comprising one or more cytokines selected from the group consisting of 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. (Item 77) 77. The kit of any one of items 68 to 76, comprising an agonist of a receptor selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain-containing 2 (NOD2). (Item 78) 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), and V-set immunoregulatory receptor (VSIR, B7 H5, 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 containing Ig and ITIM domains (TIGIT), and lymphocyte activation 3 (LAG 3, CD223), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), galectin 9 (LGALS9), killer-cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), killer-cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), and killer-cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). (Item 79) CD27, CD70, CD40, CD40LG, inducible T cell costimulator (ICOS, CD278), inducible T cell costimulator ligand (ICOSLG, B7H2), TNF receptor superfamily member 4 (TNFRSF4, OX40), TNF superfamily member 4 (TNFSF4, OX40L), TNFRSF9 (CD137), TNFSF9 (CD137L), and TNFRSF18 (GITR) 79. The kit of any one of Items 68 to 78, comprising an agonist, activator, or stimulator of a T cell stimulatory immune checkpoint protein or receptor selected from the group consisting of TNFSF18 (GITRL), CD80 (B7-1), CD28, nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), and poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). (Item 80) the CTLA4 inhibitor is 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, 79. The kit of item 78, wherein the antibody is selected from the group consisting of 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. (Item 81) The PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, AB122 (zimbarelimab), 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, B I-754091, AGEN-2034(), JS-001 (toripalimab), JNJ-63723283, genolimu- zumab (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 (dostallimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WB P-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 / TGFβ-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. (Item 82) 82. The kit according to any one of items 68 to 81, further comprising administering to the subject an inhibitor of CD47. (Item 83) The kit according to any one of Items 68 to 82, further comprising one or more antiviral agents. (Item 84) 84. The kit of item 83, wherein the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors. (Item 85) 54. A method for inducing an immune response against human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject one or more of the fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, or one or more immunogenic compositions according to any one of items 40 to 45, or one or more pharmaceutical compositions according to any one of items 46 to 54. (Item 86) 54. A method for treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof, comprising administering to the subject one or more of the fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, or one or more immunogenic compositions according to any one of items 40 to 45, or one or more pharmaceutical compositions according to any one of items 46 to 54. (Item 87) 87. The method of claim 85 or 86, comprising administering a single fusion polypeptide, or a polynucleotide or viral expression vector encoding said fusion polypeptide, wherein said fusion polypeptide comprises two or more multivalent polypeptide segments, e.g., bivalent polypeptide segments. (Item 88) 87. The method of item 85 or 86, wherein two or more fusion polypeptides, or two or more viral expression vectors encoding said fusion polypeptides, are administered to the subject simultaneously or in parallel. (Item 89) 89. The method according to any one of items 85 to 88, wherein the two or more fusion polypeptides, or the two or more polynucleotides, or the two or more viral expression vectors encoding said fusion polypeptides, are in the form of a bivalent antigen composition. (Item 90) 90. The method of any one of Items 85 to 89, comprising administering to the subject one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, and 430 to 435, 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 to 377, 407 to 411, 422 to 424, and 430 to 435. (Item 91) 91. The method of any one of Items 85 to 90, comprising administering to the subject one or more adenoviral vectors, wherein each adenoviral vector comprises one or more polynucleotides encoding one or more fusion proteins comprising the amino acid sequence of any one of SEQ ID NOs: 345 to 377, 407 to 411, 422 to 424, and 430 to 435, 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 to 377, 407 to 411, 422 to 424, and 430 to 435. (Item 92) administering to the subject one or more viral vectors, each viral vector comprising: SEQ ID NOs: 345 and 346, SEQ ID NOs: 347 and 348, SEQ ID NOs: 349 and 350, SEQ ID NOs: 351 and 352, SEQ ID NOs: 430 and 352, SEQ ID NOs: 357 and 358, SEQ ID NOs: 360 and 362, SEQ ID NOs: 359 and 361, SEQ ID NOs: 351 and 357, SEQ ID NOs: 351 and 358, SEQ ID NOs: 351 and 359, SEQ ID NOs: 351 and 360, SEQ ID NOs: 351 and 361, SEQ ID NOs: 351 and 362, SEQ ID NOs: 351 and 407, SEQ ID NOs: 351 and 408, SEQ ID NOs: 351 and 409, SEQ ID NOs: 351 and 410, SEQ ID NOs: 352 and 357, SEQ ID NOs: 352 and 358, SEQ ID NOs: 352 and 359, SEQ ID NOs: 352 and 360, SEQ ID NOs: 352 and 361, SEQ ID NOs: 352 and 362, SEQ ID NOs: 352 and 407, SEQ ID NOs: 352 and 408, SEQ ID NOs: 352 and 409, SEQ ID NOs: 352 and 410, SEQ ID NOs: 430 and 357, SEQ ID NOs: 430 and 358, SEQ ID NOs: 430 and 359, SEQ ID NOs: 430 and 360, SEQ ID NOs: 430 and 361, SEQ ID NOs: 430 and 362, SEQ ID NOs: 407 and 409, SEQ ID NOs: 407 and 408, SEQ ID NOs: 408 and 410, or 92. The method according to any one of items 85 to 91, comprising 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 NOs: 409 and 410. (Item 93) To the subject, 1) one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 351 to 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 to 356 and 430; 2) The method according to any one of Items 85 to 91, comprising administering one or more fusion polypeptides, or polynucleotides encoding the fusion polypeptides, or viral expression vectors expressing the fusion polypeptides, wherein the fusion polypeptides comprise or consist of an amino acid sequence of any one of SEQ ID NOs: 357 to 366 and 407 to 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 to 366 and 407 to 410. (Item 94) 93. The method of any one of items 85 to 92, wherein the subject is infected with, suspected of being infected with, or at risk of being infected with HIV-1. (Item 95) 95. The method of any one of items 85 to 94, wherein the subject is chronically infected with HIV-1. (Item 96) 96. The method of any one of items 85 to 95, wherein the subject is acutely infected with HIV-1. (Item 97) 97. The method of any one of items 85 to 96, wherein the subject has HIV-1 infection at Fiebig stage IV or earlier, such as Fiebig stage III, Fiebig stage II or Fiebig stage I. (Item 98) 98. The method of any one of items 85 to 97, wherein the composition is administered via a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., oral, intranasal, rectal, intravaginal). (Item 99) per dose, approximately 10 3 ~about 10 15 Viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (vp), for example, about 10 4 ~about 10 7 Viral FFU or PFU or IU or vp, for example, about 10 3 ~about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 99. The method of any one of items 85 to 98, comprising administering viral FFU or PFU or IU or vp. (Item 100) A prime-boost regimen comprising: (i) 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.); or (ii) The method of any one of paragraphs 85 to 99, comprising a prime-boost regimen comprising one or more repeats (e.g., prime-boost-prime-boost, etc.) of administering a priming composition at a first time point and a boosting composition at a second time point. (Item 101) 101. The method of claim 100, wherein the administration of the priming composition and the one or more boosting compositions is spaced at least 1 week, 2 weeks, 3 weeks, or 1 month apart, e.g., at least 2, 3, 4, 5, or 6 months apart. (Item 102) 102. The method of item 100 or 101, wherein the priming composition and the boosting composition comprise the same immunogenic composition. (Item 103) 102. The method of item 100 or 101, wherein the priming composition and the boosting composition comprise different immunogenic compositions. (Item 104) 102. The method of item 100 or 101, wherein the priming composition and the boosting composition comprise the same one or more fusion polypeptides and the same polynucleotide or viral expression vector. (Item 105) 102. The method of item 100 or 101, wherein the priming composition and the boosting composition comprise different fusion polypeptides and / or different polynucleotides or viral expression vectors. (Item 106) 106. The method of claim 105, comprising priming with a first polynucleotide or viral expression vector and boosting with a second polynucleotide or viral expression vector. (Item 107) The prime-boost regimen comprises: a) priming with a viral expression vector and boosting with a polynucleotide, wherein the polynucleotide is DNA, cDNA, mRNA, or self-replicating RNA; b) priming with a polynucleotide that is DNA, cDNA, mRNA, or self-replicating RNA and boosting with a viral expression vector; c) 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 family; d) 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 family; e) priming with a first defective attenuated viral expression vector and boosting with a second replicating attenuated viral expression vector, wherein the first and second replicating attenuated viral expression vectors are from the same, related, or unrelated taxonomic family; f) priming with a replication-deficient viral expression vector and boosting with a replication-attenuated viral expression vector; g) priming with a replication-attenuated viral expression vector and boosting with a replication-deficient viral expression vector; h) priming with a viral expression vector for lymphocytic choriomeningitis mammarenavirus (LCMV) and boosting with a viral expression vector for Pichinde mammarenavirus; i) priming with a viral expression vector of Pichinde Mammarenavirus and boosting with a viral expression vector of Lymphocytic Choriomeningitis Mammarenavirus (LCMV); j) priming with an arenavirus viral expression vector and boosting with an adenovirus viral expression vector; or k) The method of any one of items 100 to 106, comprising priming with an adenovirus viral expression vector and boosting with an arenavirus viral expression vector. (Item 108) 108. The method of any one of items 85 to 107, wherein the subject is not receiving antiretroviral therapy (ART) or ART is discontinued prior to administration of the one or more compositions. (Item 109) 109. The method of any one of items 85 to 108, wherein ART is discontinued after one or more administrations of the composition. (Item 110) 109. The method of any one of items 85 to 109, further comprising administering to the subject one or more additional therapeutic agents, for example, two, three, four, or more additional therapeutic agents. (Item 111) 111. The method of claim 110, comprising co-administering one or more agents that activate latent HIV, such as one or more latent reactivating agents (LRAs). (Item 112) 112. The method of claim 110 or 111, wherein the one or more LRAs are selected from the group consisting of one or more toll-like receptor (TLR) agonists or activators, histone deacetylase (HDAC) inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, Smyd2 inhibitors, BET-bromodomain 4 (BRD4) inhibitors, ionomycin, inhibitors of apoptosis protein (IAP) antagonists, and a second mitochondria-derived activator of caspases (SMAC) mimetic. (Item 113) 113. The method of any one of items 110 to 112, comprising co-administering one or more agonists or activators of one or more Toll-like receptors (TLRs). (Item 114) 114. The method of claim 113, wherein the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. (Item 115) 115. The method of item 113 or 114, wherein the TLR7 agonist is selected from the group consisting of GS9620 (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). (Item 116) 116. The method of any one of paragraphs 110 to 115, comprising co-administering one or more interleukin receptor agonists of interleukins selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, GM-CSF, and FLT3LG. (Item 117) 117. The method of claim 116, comprising co-administering one or more cytokines selected from the group consisting of 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. (Item 118) 118. The method of any one of paragraphs 110 to 117, comprising co-administering an agonist of a receptor selected from the group consisting of fms-related tyrosine kinase 3 (FLT3), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain-containing 2 (NOD2). (Item 119) 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), and 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 containing Ig and ITIM domains (TIGIT), and lymphocyte activation 3 (LAG3, CD 223), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), galectin 9 (LGALS9), killer-cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2), killer-cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3), and killer-cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). (Item 120) CD27, CD70, CD40, CD40LG, inducible T cell costimulator (ICOS, CD278), inducible T cell costimulator ligand (ICOSLG, B7H2), TNF receptor superfamily member 4 (TNFRSF4, OX40), TNF superfamily member 4 (TNFSF4, OX40L), TNFRSF9 (CD137), TNFSF9 (CD137L), TNFRSF18 (GITR), TNFS 120. The method of any one of items 110 to 119, comprising co-administering an agonist, activator or stimulator of a T cell stimulatory immune checkpoint protein or receptor selected from the group consisting of F18 (GITRL), CD80 (B7-1), CD28, nectin cell adhesion molecule 2 (NECTIN2, CD112), CD226 (DNAM-1), and poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). (Item 121) the CTLA4 inhibitor is 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, 120. The method of item 119, wherein the antibody is selected from the group consisting of 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. (Item 122) The PD-L1 (CD274) or PD-1 (PDCD1) inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, AB122 (zimbarelimab), 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, genolimuzumab (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 (dostallimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-100 1 (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 (CT LA4 / 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-57 52 (CTLA4 / PD-1), RO-7121661 (PD-1 / TIM-3), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFβ-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. (Item 123) 123. The method of any one of items 110 to 122, further comprising administering to the subject an inhibitor of CD47. (Item 124) 124. The method of any one of items 110 to 123, further comprising administering to the subject one or more antiviral agents. (Item 125) 125. The method of claim 124, wherein the one or more antiviral agents are selected from the group consisting of HIV protease inhibitors, HIV reverse transcriptase inhibitors, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry (fusion) inhibitors, HIV maturation inhibitors, and capsid inhibitors. (Item 126) 126. The method of any one of paragraphs 85-125, wherein after one or more administrations of one or more of the compositions, optionally in combination with one or more additional therapeutic agents, the subject does not exhibit symptoms of HIV or AIDS for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or more, in the absence of antiretroviral therapy (ART). (Item 127) 127. The method of any one of items 85-126, wherein after one or more administrations of one or more of the compositions, optionally in combination with one or more additional therapeutic agents, the subject has a viral load of less than 500, e.g., 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 more, in the absence of antiretroviral therapy (ART). (Item 128) 54. One or more fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, or one or more immunogenic compositions according to any one of items 40 to 45, or one or more pharmaceutical compositions according to any one of items 46 to 54, for use in inducing an immune response against human immunodeficiency virus (HIV) in a subject in need thereof. (Item 129) 54. One or more fusion polypeptides according to any one of items 1 to 9, or one or more polynucleotides according to any one of items 10 to 13, or one or more vectors according to any one of items 18 to 33, or one or more immunogenic compositions according to any one of items 40 to 45, or one or more pharmaceutical compositions according to any one of items 46 to 54, for use in treating or preventing human immunodeficiency virus (HIV) in a subject in need thereof. [Brief explanation of the drawings]

[0293] [Figure 1] FIG. 1 shows an eight-step workflow for designing fusion polypeptides to induce antiviral responses.

[0294] [Figure 2] A representative methodology for a population-based vaccine development approach is presented.

[0295] [Figure 3] 1 illustrates the steps of the Conserved Gait Analysis (CWA) algorithm described herein.

[0296] [Figure 4A]Figure 4A shows how "bivalent conservation" can be determined based on the prevalence of the two most common 9-mers among all considered viral sequences in a population. Figure 4A discloses, in order of appearance, SEQ ID NOS: 475-476, 476-477, 476, 476, 478-479, and 479-480 in the top panel, respectively. Figure 4A also discloses "QNLQGQMVH" as SEQ ID NOS: 481, "QNIQGQMVH" as SEQ ID NOS: 482, and "PNIQGQMVH" as SEQ ID NOS: 483 in the bottom panel. [Figure 4B] Figure 4B shows how conserved regions are identified based on the "bivalent conserved" distribution across 9-mer positions. HIV-1 Gag p24 was used as a representative protein.

[0297] [Figure 5A] Figure 5A shows unique 9-mers extracted from aligned native sequences. Figures 5A-5C disclose "IIIIIIIIR" as SEQ ID NO:467, "GIIIIIIIIH" as SEQ ID NO:473, "AIIIIIIIIIK" as SEQ ID NO:474, "GIIIIIIIIR" as SEQ ID NO:484, "GIIIIIIII" as SEQ ID NO:485, "AIIIIIIIII" as SEQ ID NO:486, "IIIIIIIIK" as SEQ ID NO:487, and "IIIIIIIIIH" as SEQ ID NO:488. [Figure 5B] Figure 5B shows a directed acyclic graph constructed based on the 9-mer pair nodes and their connections. Figures 5A-5C disclose "IIIIIIIIR" as SEQ ID NO: 467, "GIIIIIIIIH" as SEQ ID NO: 473, "AIIIIIIIIIK" as SEQ ID NO: 474, "GIIIIIIIIR" as SEQ ID NO: 484, "GIIIIIIII" as SEQ ID NO: 485, "AIIIIIIIII" as SEQ ID NO: 486, "IIIIIIIIK" as SEQ ID NO: 487, and "IIIIIIIIIH" as SEQ ID NO: 488. [Figure 5C]Figure 5C shows how the 9-mers of a connected 9-mer pair are connected. When there are two options for connection, the final connection is determined by the prevalence of each connection in naturally occurring sequences. Figures 5A-5C disclose "IIIIIIIIR" as SEQ ID NO: 467, "GIIIIIIIIH" as SEQ ID NO: 473, "AIIIIIIIIIK" as SEQ ID NO: 474, "GIIIIIIIIR" as SEQ ID NO: 484, "GIIIIIIII" as SEQ ID NO: 485, "AIIIIIIIII" as SEQ ID NO: 486, "IIIIIIIIK" as SEQ ID NO: 487, and "IIIIIIIIIH" as SEQ ID NO: 488.

[0298] [Figure 6] Figure 6 shows inter- and intra-patient diversity viral sequence analysis. The first column lists SEQ ID NOs: 475-476, 476-477, 476, 476, 478-479, 479-480, and 489, and the second column lists SEQ ID NOs: 475, 490, 490, 490, 475, 475, 475, 475, 475, 489, and 491, in order of appearance, respectively.

[0299] [Figure 7] Figure 1 shows intra-patient diversity analysis of HIV-1 proteins. Bivalent vaccine sequences are quasi-species mismatched at 21-48% of positions in highly conserved regions.

[0300] [Figure 8] Figure 8 shows the results of a cross-recognition analysis of the human proteome. Figure 8 discloses, in order of appearance, the "human protein 9mer" sequence as SEQ ID NO: 492 and the "HIV peptide" sequences as SEQ ID NOs: 493-498 and 493, respectively.

[0301] [Figure 9] Polypeptide segment alignment analysis demonstrates how the possible presentation of harmful or undesirable epitopes within junction regions can be reduced or eliminated.

[0302] [Figure 10A] Figure 10A shows the basic methodology of the "short peptide" approach described in Example 3, where a set of HLA-restricted 9-mers are selected from bivalent constructs and combined to form an HLA-restricted vaccine construct. [Figure 10B] Figure 10B shows the basic methodology of the "long peptide" approach described in Example 3, where a set of HLA-restricted 9-mers are selected from the bivalent constructs and combined to form an HLA-restricted vaccine construct.

[0303] [Figure 11] We demonstrate the incorporation of considerations of binding to MHC class I molecules, including pan-allelic (e.g., HLA supertypes: A01, A02, A03, A24, B07, B08, B27, B44, B58, B62) and allele-specific (e.g., A*0201 as a representative human MHC class I allele) analysis of identified conserved viral protein regions.

[0304] [Figure 12A] Figure 12A shows how conserved region positions are classified into four categories. Antiviral vaccine design approaches can be improved by incorporating deep sequencing analysis and MHC class I binding data. Figure 12A discloses SEQ ID NOS: 499-501, 121, 502-505, 499-501, 121, and 502-510, respectively, in order of appearance. [Figure 12B] FIG. 12B shows an approach to improve current antiviral vaccine design approaches by incorporating deep sequencing analysis and MHC class I binding data into within-patient sequence analysis.

[0305] [Figure 13] Deep sequencing data and patient HLA data analysis are included in the approach to create personalized vaccine constructs.

[0306] [Figure 14]To further improve the HLA-restricted vaccine constructs described in Example 3, an approach involving deep sequencing data analysis is presented.

[0307] [Figure 15] We present a rational approach to antiviral immunogen design that considers inter- and intra-patient viral sequence diversity, host MHC class I and class II molecule binding, and T cell recognition. The approach and resulting immunogens are exemplified herein by immunogens that induce human immune responses against HIV-1.

[0308] [Figure 16]

[0039] Figure 16 shows the polypeptide segments encoded by the HIV-1 Env gene used in the fusion polypeptide constructs described herein. The Env HIV-1 HXB2 reference polypeptide (SEQ ID NO: 403) sequence is underlined. Figure 16 also discloses, in order of appearance, SEQ ID NOs: 1, 10, 4, 15, 6, 19, 21, 27-28, 30, 37, 511, 512, and 60, respectively.

[0309] [Figure 17]

[0033] Figure 17 shows polypeptide segments encoded by the HIV-1 Gag gene used in the fusion polypeptide constructs described herein. The Gag HIV-1 HXB2 reference polypeptide (SEQ ID NO: 404) sequence is underlined. Figure 17 also discloses, in order of appearance, SEQ ID NOs: 70, 76, 78, 87, 94, 96-97, 99, 339, 107, 341, 117, 113, 119, 121, 123, and 137, respectively.

[0310] [Figure 18]

[0023] Figure 18 shows polypeptide segments encoded by the HIV-1 Nef gene used in the fusion polypeptide constructs described herein. The Nef HIV-1 HXB2 reference polypeptide having a tryptophan (W) at position 124 (SEQ ID NO: 405) is underlined. Figure 18 also discloses SEQ ID NOs: 151, 513, 153, 514, 165, 515, and 172, respectively, in order of appearance.

[0311] [Figure 19A]

[0023] Figure 1 shows the polypeptide segments encoded by the HIV-1 Pol genes used in the fusion polypeptide constructs described herein. The Pol HIV-1 HXB2 reference polypeptide (SEQ ID NO: 406) sequence is underlined. 19A-19C also disclose SEQ ID NOs: 176, 188, 181, 190, 192, 516, 209, 517, 197, 210, 201, 211, 213, 518, 217, 219, 223, 222, 225, 227, 229-230, 232, 234, 236, 238, 240-241, 243, 259, 261, 265, 274, 282, 276, 294, 296, 300, 298, 302-303, 305, 519, 311, 319, 322-323, 334, 325, 336, 329, 327, 331, and 333, respectively. [Figure 19B]

[0023] Figure 1 shows the polypeptide segments encoded by the HIV-1 Pol genes used in the fusion polypeptide constructs described herein. The Pol HIV-1 HXB2 reference polypeptide (SEQ ID NO: 406) sequence is underlined. 19A-19C also disclose SEQ ID NOs: 176, 188, 181, 190, 192, 516, 209, 517, 197, 210, 201, 211, 213, 518, 217, 219, 223, 222, 225, 227, 229-230, 232, 234, 236, 238, 240-241, 243, 259, 261, 265, 274, 282, 276, 294, 296, 300, 298, 302-303, 305, 519, 311, 319, 322-323, 334, 325, 336, 329, 327, 331, and 333, respectively. [Figure 19C]

[0023] Figure 1 shows the polypeptide segments encoded by the HIV-1 Pol genes used in the fusion polypeptide constructs described herein. The Pol HIV-1 HXB2 reference polypeptide (SEQ ID NO: 406) sequence is underlined. 19A-19C also disclose SEQ ID NOs: 176, 188, 181, 190, 192, 516, 209, 517, 197, 210, 201, 211, 213, 518, 217, 219, 223, 222, 225, 227, 229-230, 232, 234, 236, 238, 240-241, 243, 259, 261, 265, 274, 282, 276, 294, 296, 300, 298, 302-303, 305, 519, 311, 319, 322-323, 334, 325, 336, 329, 327, 331, and 333, respectively.

[0312] [Figure 20] This example shows a modified vaccine expression cassette for expressing a fusion polypeptide in an adenovirus expression vector under the control of a CMV promoter. To determine approaches for combining conserved regions, candidate viral vector vaccines were constructed for the expression of polypeptide segments from computationally defined conserved regions, as well as regions combined as (A) a fusion polypeptide construct (SEQ ID NOs: 345 / 346), (B) a processing spacer containing an F2A proteolytic cleavage site (SEQ ID NOs: 349 / 350), (C) a flexible linker (e.g., AAA (SEQ ID NO: 378)) (SEQ ID NOs: 347 / 348), (D) a fusion polypeptide containing only the p17 and p24 conserved regions, (E) a fusion polypeptide containing only the protease, RT, and integrase conserved regions, and (F) an NEF-only construct (SEQ ID NOs: 151 / 152).

[0313] [Figure 21A](A) Plasmid DNA containing vaccine immunogens (fusion, F2A cleavage site, AAA linker (SEQ ID NO: 378)) or fusion segments (e.g., Pol PR-RT), each representing a different conjugation strategy, was transfected into Expi293™ cells. Transfection efficiency was determined by assessing % GFP expression in transfected Expi293F cells by flow cytometry. Results shown are representative of multiple independent experiments. (B) All plasmid DNAs contained p24 in the expression cassette. Expression efficiency was assessed by p24 ELISA. (C) Translation products of vaccine transgene constructs were analyzed. The identity of the translated polypeptides was confirmed by Western blot immunoprecipitation with anti-Nef antibody (2 μg / mL, 0.5 s exposure). The top band corresponded to the expected full-length translation product (88 kDa) in the fusion and AAA linker (SEQ ID NO: 378)-containing constructs. The F2A-containing translation product was not detected by anti-Nef antibody, indicating cleavage of the Nef product due to the absence of the full-length construct. As a control for equal loading, membranes were probed with an antibody against α-tubulin. [Figure 21B](A) Plasmid DNA containing vaccine immunogens (fusion, F2A cleavage site, AAA linker (SEQ ID NO: 378)) or fusion segments (e.g., Pol PR-RT), each representing a different conjugation strategy, was transfected into Expi293™ cells. Transfection efficiency was determined by assessing % GFP expression in transfected Expi293F cells by flow cytometry. Results shown are representative of multiple independent experiments. (B) All plasmid DNAs contained p24 in the expression cassette. Expression efficiency was assessed by p24 ELISA. (C) Translation products of vaccine transgene constructs were analyzed. The identity of the translated polypeptides was confirmed by Western blot immunoprecipitation with anti-Nef antibody (2 μg / mL, 0.5 s exposure). The top band corresponded to the expected full-length translation product (88 kDa) in the fusion and AAA linker (SEQ ID NO: 378)-containing constructs. The F2A-containing translation product was not detected by anti-Nef antibody, indicating cleavage of the Nef product due to the absence of the full-length construct. As a control for equal loading, membranes were probed with an antibody against α-tubulin. [Figure 21C](A) Plasmid DNA containing vaccine immunogens (fusion, F2A cleavage site, AAA linker (SEQ ID NO: 378)) or fusion segments (e.g., Pol PR-RT), each representing a different conjugation strategy, was transfected into Expi293™ cells. Transfection efficiency was determined by assessing % GFP expression in transfected Expi293F cells by flow cytometry. Results shown are representative of multiple independent experiments. (B) All plasmid DNAs contained p24 in the expression cassette. Expression efficiency was assessed by p24 ELISA. (C) Translation products of vaccine transgene constructs were analyzed. The identity of the translated polypeptides was confirmed by Western blot immunoprecipitation with anti-Nef antibody (2 μg / mL, 0.5 s exposure). The top band corresponded to the expected full-length translation product (88 kDa) in the fusion and AAA linker (SEQ ID NO: 378)-containing constructs. The F2A-containing translation product was not detected by anti-Nef antibody, indicating cleavage of the Nef product due to the absence of the full-length construct. As a control for equal loading, membranes were probed with an antibody against α-tubulin.

[0314] [Figure 22] Comparison of the differentiation phenotype of immature and mature monocyte-derived dendritic cells (mMoDCs) is shown. Monocyte-derived DCs (MoDCs) were matured for 8 days in the presence of cytokines and analyzed by flow cytometry for the expression of CD11c, HLA-DR, CD14, CD430, DCSIGN, CD83, CD86, and OX40L.

[0315] [Figure 23]Representative moDC transduction efficiencies using GFP-expressing Ad5 / 35 viral vectors at a multiplicity of infection (MOI) of 1000 PFU in eight human donors are shown on day 3 post-transfection. The percentage of cells expressing GFP by flow cytometry is shown on the y-axis. The x-axis represents vaccine immunogen constructs consisting of a full-length Gag-Nef immunogen designed using the conserved region (SEQ ID NO: 428) in p17-p24 only and each of three fusion approaches: F2A (SEQ ID NO: 347), fusion (SEQ ID NO: 349), and AAA linker (SEQ ID NO: 345) ("AAA" disclosed as SEQ ID NO: 378). The amino acid sequences are shown in Table 1.

[0316] [Figure 24A] Figure 1 shows the priming of antigen-specific T cells by vaccine constructs expressing conserved regions of HIV-1 linked or connected by a fusion, F2A proteolytic cleavage sequence, or AAA linker (SEQ ID NO: 378). (A) The extent of primed responses assessed by IFN-γ ELISpot assay 10 days after coculture of PBMCs with vaccine vector-transduced autologous moDCs. PBMCs derived from HIV-infected donors with pre-existing HIV-specific responses at baseline. (B) The breadth of the response, defined as the number of de novo recognized peptide pools (excluding pre-existing baseline responses). No statistical significance was observed between the different groups in this analysis. [Figure 24B] Figure 1 shows the priming of antigen-specific T cells by vaccine constructs expressing conserved regions of HIV-1 linked or connected by a fusion, F2A proteolytic cleavage sequence, or AAA linker (SEQ ID NO: 378). (A) The extent of primed responses assessed by IFN-γ ELISpot assay 10 days after coculture of PBMCs with vaccine vector-transduced autologous moDCs. PBMCs derived from HIV-infected donors with pre-existing HIV-specific responses at baseline. (B) The breadth of the response, defined as the number of de novo recognized peptide pools (excluding pre-existing baseline responses). No statistical significance was observed between the different groups in this analysis.

[0317] [Figure 25A] Figure 1 shows in vitro priming of antigen-specific T cells from HIV-infected donors. Exemplary flow cytometry plots show IFN-γ production by intracellular cytokine staining (ICS) 10 days after co-culture of PBMCs with vaccine vector-transduced autologous moDCs. The x-axis indicates the vaccine construct used for in vitro priming. Each bar represents stimulation with a peptide pool from Gag p17, Gag p24, integrase, Pol (protease / RT), and Nef. The percentage of IFN-γ+ T cells indicates (A) CD8+ T cell responses and (B) CD4+ T cells. (●) Gag p17, (■) Gag p24, (▲) INT, (▼) Pol, [ka] Nef. In this analysis, no statistical significance was observed between the different groups. Figures 25A-B disclose "AAA" as SEQ ID NO: 378. [Figure 25B] Figure 1 shows in vitro priming of antigen-specific T cells from HIV-infected donors. Exemplary flow cytometry plots show IFN-γ production by intracellular cytokine staining (ICS) 10 days after co-culture of PBMCs with vaccine vector-transduced autologous moDCs. The x-axis indicates the vaccine construct used for in vitro priming. Each bar represents stimulation with a peptide pool from Gag p17, Gag p24, integrase, Pol (protease / RT), and Nef. The percentage of IFN-γ+ T cells indicates (A) CD8+ T cell responses and (B) CD4+ T cells. (●) Gag p17, (■) Gag p24, (▲) INT, (▼) Pol, [ka] Nef. In this analysis, no statistical significance was observed between the different groups. Figures 25A-B disclose "AAA" as SEQ ID NO: 378.

[0318] [Figure 26A]1 shows in vitro priming of antigen-specific T cells from viraemic and virally infected HIV-1 infected donors. Representative figures show IFN-γ production by intracellular cytokine staining (ICS) on days 7 (first round) and 14 (second round) after co-culture of PBMCs with autologous moDCs transduced with adenoviral vectors containing Gag-Nef immunogens designed according to one of three fusion approaches (F2A (SEQ ID NO: 347), fusion (SEQ ID NO: 349) and AAA linker (SEQ ID NO: 345 "AAA", disclosed as SEQ ID NO: 378) in viremic (A) and virus-infected (B) donors. The x-axis indicates the vaccine construct used for in vitro priming. Each bar represents stimulation with peptide pools from Gag p17, Gag p24, integrase, Pol (protease / RT), and Nef. The percentage of IFN-γ+ T cells indicates (A) CD8+ T cell responses, (B) CD4+ T cells. (●) Gag p17, (■) Gag p24, (▲) INT, (▼) Pol, [ka] This is Nef. [Figure 26B]1 shows in vitro priming of antigen-specific T cells from viraemic and virally infected HIV-1 infected donors. Representative figures show IFN-γ production by intracellular cytokine staining (ICS) on days 7 (first round) and 14 (second round) after co-culture of PBMCs with autologous moDCs transduced with adenoviral vectors containing Gag-Nef immunogens designed according to one of three fusion approaches (F2A (SEQ ID NO: 347), fusion (SEQ ID NO: 349) and AAA linker (SEQ ID NO: 345 "AAA", disclosed as SEQ ID NO: 378) in viremic (A) and virus-infected (B) donors. The x-axis indicates the vaccine construct used for in vitro priming. Each bar represents stimulation with peptide pools from Gag p17, Gag p24, integrase, Pol (protease / RT), and Nef. The percentage of IFN-γ+ T cells indicates (A) CD8+ T cell responses, (B) CD4+ T cells. (●) Gag p17, (■) Gag p24, (▲) INT, (▼) Pol, [ka] This is Nef.

[0319] [Figure 27] Figure 1 shows antigen functional signature profiling of antigen-specific CD8+ and CD4+ T cells primed using moDCs transduced with vectors containing immunogen expression cassettes with fusion-linked conserved regions, F2A proteolytic cleavage sequences, or AAA linkers (SEQ ID NO: 378). Pie charts show the adjusted polyfunctional (subtracting background responses) characteristics of primed CD8+ or CD4+ T cells (one to four functions—CD107a, IFN-γ, TNF-α, and IL-2).

[0320] [Figure 28A]Viral vectors expressing HIV-1 antigens that induce a high degree of CD8+ T cell response after immunization are shown. (A) Immunization and sampling schedule. Groups of Balb / c mice were immunized with Ad5 / 35 vectors expressing fusion-linked HIV-1 conserved region sequences, the F2A proteolytic cleavage sequence, or a flexible AAA linker (SEQ ID NO: 378). Mice were immunized using a homologous prime-boost schedule on days 1 and 29, and each group was analyzed on days 16 (prime) or 36 (prime-boost). (B) Immunogenicity on day 16 and (C) 36 post-immunization was determined by assessing the frequency of ex vivo peptide-specific splenocytes using an IFN-γ ELISPOT assay to detect IFN-γ-producing cells. A set of 15-mer peptides matching 11 amino acids and overlapping the F2A peptide within the vaccine construct was synthesized and used to stimulate splenocytes in ELISpot and ICS assays. [Figure 28B] Viral vectors expressing HIV-1 antigens that induce a high degree of CD8+ T cell response after immunization are shown. (A) Immunization and sampling schedule. Groups of Balb / c mice were immunized with Ad5 / 35 vectors expressing fusion-linked HIV-1 conserved region sequences, the F2A proteolytic cleavage sequence, or a flexible AAA linker (SEQ ID NO: 378). Mice were immunized using a homologous prime-boost schedule on days 1 and 29, and each group was analyzed on days 16 (prime) or 36 (prime-boost). (B) Immunogenicity on day 16 and (C) 36 post-immunization was determined by assessing the frequency of ex vivo peptide-specific splenocytes using an IFN-γ ELISPOT assay to detect IFN-γ-producing cells. A set of 15-mer peptides matching 11 amino acids and overlapping the F2A peptide within the vaccine construct was synthesized and used to stimulate splenocytes in ELISpot and ICS assays. [Figure 28C]Viral vectors expressing HIV-1 antigens that induce a high degree of CD8+ T cell response after immunization are shown. (A) Immunization and sampling schedule. Groups of Balb / c mice were immunized with Ad5 / 35 vectors expressing fusion-linked HIV-1 conserved region sequences, the F2A proteolytic cleavage sequence, or a flexible AAA linker (SEQ ID NO: 378). Mice were immunized using a homologous prime-boost schedule on days 1 and 29, and each group was analyzed on days 16 (prime) or 36 (prime-boost). (B) Immunogenicity on day 16 and (C) 36 post-immunization was determined by assessing the frequency of ex vivo peptide-specific splenocytes using an IFN-γ ELISPOT assay to detect IFN-γ-producing cells. A set of 15-mer peptides matching 11 amino acids and overlapping the F2A peptide within the vaccine construct was synthesized and used to stimulate splenocytes in ELISpot and ICS assays.

[0321] [Figure 29A] Figure 29 shows functional profiles of vaccine-induced CD8+ T cell responses in Balb / c (A) and C57 BL / 6 animals (B). Flow cytometry profiles of CD8+ T cells capable of mediating degranulation (CD107a), IFN-γ, IL-2, and TNF-α production were analyzed to demonstrate the functional composition of responses for animals in each vaccination group. Splenocytes were stimulated with the relevant peptide pool (p24, as shown herein) for 6 hours and stained as described in the Methods. Pie charts summarize the data, with each slice of the pie corresponding to the proportion of CD8+ T cells with a given number of functions within the total CD8+ T cell population. All possible combinations of responses are shown on the x-axis, and the percentage of functionally distinct CD8+ T cells within the total population is shown on the y-axis. Means and SDs are shown. Figures 29A-B disclose "AAA" as SEQ ID NO: 378. [Figure 29B]Figure 29 shows functional profiles of vaccine-induced CD8+ T cell responses in Balb / c (A) and C57 BL / 6 animals (B). Flow cytometry profiles of CD8+ T cells capable of mediating degranulation (CD107a), IFN-γ, IL-2, and TNF-α production were analyzed to demonstrate the functional composition of responses for animals in each vaccination group. Splenocytes were stimulated with the relevant peptide pool (p24, as shown herein) for 6 hours and stained as described in the Methods. Pie charts summarize the data, with each slice of the pie corresponding to the proportion of CD8+ T cells with a given number of functions within the total CD8+ T cell population. All possible combinations of responses are shown on the x-axis, and the percentage of functionally distinct CD8+ T cells within the total population is shown on the y-axis. Means and SDs are shown. Figures 29A-B disclose "AAA" as SEQ ID NO: 378.

[0322] [Figure 30A] Figure 1 shows the memory phenotype of IFN-γ-producing cells. (A) Flow cytometry plots showing the gating strategy for defining memory subsets and exhausted phenotypes based on CCR7, CD45RA, CD27, PD-1, and CTLA-4 expression on IFN-γ+CD8+ T cells after restimulation with 2 μg / ml of the Gag p24 peptide pool. (B) Percentages of naive (CCR7+CD45RA-), effector memory cells (CCR7-CD45RA-), and central memory cells (CCR7+CD45RA-) among IFN-γ+CD8+ and IFN-γ+CD4+ T cells after restimulation with 2 μg / ml of the Gag p24 peptide pool. [Figure 30B]Figure 1 shows the memory phenotype of IFN-γ-producing cells. (A) Flow cytometry plots showing the gating strategy for defining memory subsets and exhausted phenotypes based on CCR7, CD45RA, CD27, PD-1, and CTLA-4 expression on IFN-γ+CD8+ T cells after restimulation with 2 μg / ml of the Gag p24 peptide pool. (B) Percentages of naive (CCR7+CD45RA-), effector memory cells (CCR7-CD45RA-), and central memory cells (CCR7+CD45RA-) among IFN-γ+CD8+ and IFN-γ+CD4+ T cells after restimulation with 2 μg / ml of the Gag p24 peptide pool.

[0323] [Figure 31A] Figure 31A shows that signal sequences differentially enhance the immunogenicity of vaccine immunogens (SEQ ID NOs: 369, 370, 371, 368, 367). Figure 31B shows that HIV-1 conserved region sequences are immunogenic relative to the GM-CSF signal sequence (SEQ ID NOs: 353, 363). [Figure 31B] Figure 31A shows that signal sequences differentially enhance the immunogenicity of vaccine immunogens (SEQ ID NOs: 369, 370, 371, 368, 367). Figure 31B shows that HIV-1 conserved region sequences are immunogenic relative to the GM-CSF signal sequence (SEQ ID NOs: 353, 363).

[0324] [Figure 32A]Figure 32A shows the immunization and sampling schedule. Figure 32B shows LCMV vectors expressing HIV-1 conserved region sequences derived from the Gag-Nef fusion protein sequence without the signal sequence (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356) used to immunize groups of Balb / c mice. Figures 32C-D show the immunogenicity of vaccine immunogens with different signal sequences for Gag p24 by IFN-γ ELISpot after priming on day 7 (Figure 32C) and boosting on day 27 (Figure 32D) with LCMV replication-incompetent vectors expressing the conserved Gag-Nef in the presence or absence of MCP-3, tPA, β-catenin, and GM-CSF signal sequences. Figure 32E shows the immunogenicity of Ad5 / 35 vectors expressing the conserved Pol with or without the GMCSF signal sequence in female Balb / c mice 7 days after immunization. Each point represents one individual mouse. The mean and SD are shown. No statistical significance was observed between the different groups in this analysis. [Figure 32B]Figure 32A shows the immunization and sampling schedule. Figure 32B shows LCMV vectors expressing HIV-1 conserved region sequences derived from the Gag-Nef fusion protein sequence without the signal sequence (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356) used to immunize groups of Balb / c mice. Figures 32C-D show the immunogenicity of vaccine immunogens with different signal sequences for Gag p24 by IFN-γ ELISpot after priming on day 7 (Figure 32C) and boosting on day 27 (Figure 32D) with LCMV replication-incompetent vectors expressing the conserved Gag-Nef in the presence or absence of MCP-3, tPA, β-catenin, and GM-CSF signal sequences. Figure 32E shows the immunogenicity of Ad5 / 35 vectors expressing the conserved Pol with or without the GMCSF signal sequence in female Balb / c mice 7 days after immunization. Each point represents one individual mouse. The mean and SD are shown. No statistical significance was observed between the different groups in this analysis. [Figure 32C]Figure 32A shows the immunization and sampling schedule. Figure 32B shows LCMV vectors expressing HIV-1 conserved region sequences derived from the Gag-Nef fusion protein sequence without the signal sequence (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356) used to immunize groups of Balb / c mice. Figures 32C-D show the immunogenicity of vaccine immunogens with different signal sequences for Gag p24 by IFN-γ ELISpot after priming on day 7 (Figure 32C) and boosting on day 27 (Figure 32D) with LCMV replication-incompetent vectors expressing the conserved Gag-Nef in the presence or absence of MCP-3, tPA, β-catenin, and GM-CSF signal sequences. Figure 32E shows the immunogenicity of Ad5 / 35 vectors expressing the conserved Pol with or without the GMCSF signal sequence in female Balb / c mice 7 days after immunization. Each point represents one individual mouse. The mean and SD are shown. No statistical significance was observed between the different groups in this analysis. [Figure 32D]Figure 32A shows the immunization and sampling schedule. Figure 32B shows LCMV vectors expressing HIV-1 conserved region sequences derived from the Gag-Nef fusion protein sequence without the signal sequence (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356) used to immunize groups of Balb / c mice. Figures 32C-D show the immunogenicity of vaccine immunogens with different signal sequences for Gag p24 by IFN-γ ELISpot after priming on day 7 (Figure 32C) and boosting on day 27 (Figure 32D) with LCMV replication-incompetent vectors expressing the conserved Gag-Nef in the presence or absence of MCP-3, tPA, β-catenin, and GM-CSF signal sequences. Figure 32E shows the immunogenicity of Ad5 / 35 vectors expressing the conserved Pol with or without the GMCSF signal sequence in female Balb / c mice 7 days after immunization. Each point represents one individual mouse. The mean and SD are shown. No statistical significance was observed between the different groups in this analysis. [Figure 32E]Figure 32A shows the immunization and sampling schedule. Figure 32B shows LCMV vectors expressing HIV-1 conserved region sequences derived from the Gag-Nef fusion protein sequence without the signal sequence (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356) used to immunize groups of Balb / c mice. Figures 32C-D show the immunogenicity of vaccine immunogens with different signal sequences for Gag p24 by IFN-γ ELISpot after priming on day 7 (Figure 32C) and boosting on day 27 (Figure 32D) with LCMV replication-incompetent vectors expressing the conserved Gag-Nef in the presence or absence of MCP-3, tPA, β-catenin, and GM-CSF signal sequences. Figure 32E shows the immunogenicity of Ad5 / 35 vectors expressing the conserved Pol with or without the GMCSF signal sequence in female Balb / c mice 7 days after immunization. Each point represents one individual mouse. The mean and SD are shown. No statistical significance was observed between the different groups in this analysis.

[0325] [Figure 33A]Figure 32A shows the immunization and sampling schedule. Figure 33B shows Ad5 / 35 vectors expressing HIV-1 conserved regions derived from Gag-Nef and Pol fusion protein sequences without signal sequences (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356), which were used to immunize groups of Balb / c mice. The fusion protein sequences are provided in Table J. Figures 33C-D show the immunogenicity of vaccine immunogens with different signal sequences by flow cytometry analysis of IFN-γ ELISpot (Figure 33C) and intracellular IFN-γ CD8 T cells (Figure 33D) after primary immunization on day 16. Each point represents one individual mouse. Means and SDs are shown. Statistical significance between groups was determined using a nonparametric Mann-Whitney test. *P≦0.05, **P≦0.001 [Figure 33B] Figure 32A shows the immunization and sampling schedule. Figure 33B shows Ad5 / 35 vectors expressing HIV-1 conserved regions derived from Gag-Nef and Pol fusion protein sequences without signal sequences (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356), which were used to immunize groups of Balb / c mice. The fusion protein sequences are provided in Table J. Figures 33C-D show the immunogenicity of vaccine immunogens with different signal sequences by flow cytometry analysis of IFN-γ ELISpot (Figure 33C) and intracellular IFN-γ CD8 T cells (Figure 33D) after primary immunization on day 16. Each point represents one individual mouse. Means and SDs are shown. Statistical significance between groups was determined using a nonparametric Mann-Whitney test. *P≦0.05, **P≦0.001 [Figure 33C]Figure 32A shows the immunization and sampling schedule. Figure 33B shows Ad5 / 35 vectors expressing HIV-1 conserved regions derived from Gag-Nef and Pol fusion protein sequences without signal sequences (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356), which were used to immunize groups of Balb / c mice. The fusion protein sequences are provided in Table J. Figures 33C-D show the immunogenicity of vaccine immunogens with different signal sequences by flow cytometry analysis of IFN-γ ELISpot (Figure 33C) and intracellular IFN-γ CD8 T cells (Figure 33D) after primary immunization on day 16. Each point represents one individual mouse. Means and SDs are shown. Statistical significance between groups was determined using a nonparametric Mann-Whitney test. *P≦0.05, **P≦0.001 [Figure 33D] Figure 32A shows the immunization and sampling schedule. Figure 33B shows Ad5 / 35 vectors expressing HIV-1 conserved regions derived from Gag-Nef and Pol fusion protein sequences without signal sequences (SEQ ID NOs: 357, 430) and with signal sequences derived from GM-CSF (SEQ ID NOs: 353, 363), t-PA (SEQ ID NO: 354), MCP-3 (SEQ ID NO: 355), and β-catenin (SEQ ID NO: 356), which were used to immunize groups of Balb / c mice. The fusion protein sequences are provided in Table J. Figures 33C-D show the immunogenicity of vaccine immunogens with different signal sequences by flow cytometry analysis of IFN-γ ELISpot (Figure 33C) and intracellular IFN-γ CD8 T cells (Figure 33D) after primary immunization on day 16. Each point represents one individual mouse. Means and SDs are shown. Statistical significance between groups was determined using a nonparametric Mann-Whitney test. *P≦0.05, **P≦0.001

[0326] [Figure 34A]Figure 34 shows the immunogenicity of LCMV vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 34A depicts the immunization and sampling schedule showing time points for prime and boost vaccinations and assessment of responses by IFN-γ ELISpot. Figure 34B depicts the immunogen constructs used for vaccination with LCMV vectors expressing A*0201 sequences in the absence (SEQ ID NOs: 367, 431) or presence (SEQ ID NOs: 368, 432) of the GM-CSF signal sequence. Mice were also immunized with LCMV vectors expressing Gag-Nef and Pol fusion protein sequences in the absence (SEQ ID NO: 430 + SEQ ID NO: 357) or presence (SEQ ID NO: 353 + SEQ ID NO: 363) of the GM-CSF signal sequence. The fusion protein sequences are provided in Table J. The A*0201 sequence contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is arranged in a vector as a beaded structure. Figure 34C shows the magnitude of IFN-γ responses to the A*0201 peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. Figure 34D shows the magnitude of IFN-γ responses to the Gag peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. In Figures 34C-D, the Y-axis represents the magnitude of IFN-γ responses to specific peptide pool stimulation as the number of spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming and boosting in which peptide-specific responses were studied. No statistical significance was observed between the different groups in this analysis. [Figure 34B]Figure 34 shows the immunogenicity of LCMV vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 34A depicts the immunization and sampling schedule showing time points for prime and boost vaccinations and assessment of responses by IFN-γ ELISpot. Figure 34B depicts the immunogen constructs used for vaccination with LCMV vectors expressing A*0201 sequences in the absence (SEQ ID NOs: 367, 431) or presence (SEQ ID NOs: 368, 432) of the GM-CSF signal sequence. Mice were also immunized with LCMV vectors expressing Gag-Nef and Pol fusion protein sequences in the absence (SEQ ID NO: 430 + SEQ ID NO: 357) or presence (SEQ ID NO: 353 + SEQ ID NO: 363) of the GM-CSF signal sequence. The fusion protein sequences are provided in Table J. The A*0201 sequence contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is arranged in a vector as a beaded structure. Figure 34C shows the magnitude of IFN-γ responses to the A*0201 peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. Figure 34D shows the magnitude of IFN-γ responses to the Gag peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. In Figures 34C-D, the Y-axis represents the magnitude of IFN-γ responses to specific peptide pool stimulation as the number of spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming and boosting in which peptide-specific responses were studied. No statistical significance was observed between the different groups in this analysis. [Figure 34C]Figure 34 shows the immunogenicity of LCMV vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 34A depicts the immunization and sampling schedule showing time points for prime and boost vaccinations and assessment of responses by IFN-γ ELISpot. Figure 34B depicts the immunogen constructs used for vaccination with LCMV vectors expressing A*0201 sequences in the absence (SEQ ID NOs: 367, 431) or presence (SEQ ID NOs: 368, 432) of the GM-CSF signal sequence. Mice were also immunized with LCMV vectors expressing Gag-Nef and Pol fusion protein sequences in the absence (SEQ ID NO: 430 + SEQ ID NO: 357) or presence (SEQ ID NO: 353 + SEQ ID NO: 363) of the GM-CSF signal sequence. The fusion protein sequences are provided in Table J. The A*0201 sequence contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is arranged in a vector as a beaded structure. Figure 34C shows the magnitude of IFN-γ responses to the A*0201 peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. Figure 34D shows the magnitude of IFN-γ responses to the Gag peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. In Figures 34C-D, the Y-axis represents the magnitude of IFN-γ responses to specific peptide pool stimulation as the number of spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming and boosting in which peptide-specific responses were studied. No statistical significance was observed between the different groups in this analysis. [Figure 34D]Figure 34 shows the immunogenicity of LCMV vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 34A depicts the immunization and sampling schedule showing time points for prime and boost vaccinations and assessment of responses by IFN-γ ELISpot. Figure 34B depicts the immunogen constructs used for vaccination with LCMV vectors expressing A*0201 sequences in the absence (SEQ ID NOs: 367, 431) or presence (SEQ ID NOs: 368, 432) of the GM-CSF signal sequence. Mice were also immunized with LCMV vectors expressing Gag-Nef and Pol fusion protein sequences in the absence (SEQ ID NO: 430 + SEQ ID NO: 357) or presence (SEQ ID NO: 353 + SEQ ID NO: 363) of the GM-CSF signal sequence. The fusion protein sequences are provided in Table J. The A*0201 sequence contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is arranged in a vector as a beaded structure. Figure 34C shows the magnitude of IFN-γ responses to the A*0201 peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. Figure 34D shows the magnitude of IFN-γ responses to the Gag peptide pool from both prime-vaccinated and prime / boost-vaccinated animals. In Figures 34C-D, the Y-axis represents the magnitude of IFN-γ responses to specific peptide pool stimulation as the number of spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming and boosting in which peptide-specific responses were studied. No statistical significance was observed between the different groups in this analysis.

[0327] [Figure 35A]Figure 35A shows the immunogenicity of Ad5 / 35 vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 35A depicts the immunization and sampling schedule showing time points for prime vaccination and assessment of responses by IFN-γ ELISpot. Figure 35B shows the immunogen construct used for vaccination, in which the A*0201 sequence without the signal sequence is (SEQ ID NOs: 367, 431), the GM-CSF signal sequence is (SEQ ID NOs: 368, 432), the tPA signal sequence is (SEQ ID NOs: 369, 433), the MCP-3 signal sequence is (SEQ ID NOs: 370, 434), the β-catenin signal sequence is (SEQ ID NOs: 371, 435), and the LAMP-1 N- and C-terminal signal sequences are (SEQ ID NO: 372), and each vector expresses the GM-CSF Gag-Nef and GM-CSF Pol fusion protein sequences (SEQ ID NO: 353 + SEQ ID NO: 363), respectively. The fusion protein sequences are provided in Table J. The A*0201 sequence in the vector contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is cloned into the Ad5 / 35 vector as a bead-and-beads configuration. Figure 35C shows the extent of IFN-γ responses to the A*0201 peptide pool from prime-vaccinated animals. The Y-axis represents the extent of IFN-γ responses to A*0201 peptide pool stimulation as the number of spot-forming cells (SFC) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming. Figure 35D shows the extent of IFN-γ responses to Gag, Nef, Pol-1, and Pol-2 peptide pools in GM-CSF-Gag / Nef+GM-CSF-Pol-vaccinated animals. The vector sequence contains not only A*0201 epitope-specific sequences but also overall conserved sequences. The Y-axis represents the extent of IFN-γ responses in GM-CSF-Gag / Nef+GM-CSCF-Pol vaccine-primed animals, and the X-axis represents the specific peptide pool used for stimulation.Each bar represents stimulation with a peptide pool from Gag p24, Gag p17, Nef, Pol-1 (protease / RT), and Pol-2 (integrase) responses. Responses are expressed as spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. Statistical significance between groups was determined using the nonparametric Mann-Whitney test. *P ≤ 0.05, **P ≤ 0.001. [Figure 35B]Figure 35A shows the immunogenicity of Ad5 / 35 vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 35A depicts the immunization and sampling schedule showing time points for prime vaccination and assessment of responses by IFN-γ ELISpot. Figure 35B shows the immunogen construct used for vaccination, in which the A*0201 sequence without the signal sequence is (SEQ ID NOs: 367, 431), the GM-CSF signal sequence is (SEQ ID NOs: 368, 432), the tPA signal sequence is (SEQ ID NOs: 369, 433), the MCP-3 signal sequence is (SEQ ID NOs: 370, 434), the β-catenin signal sequence is (SEQ ID NOs: 371, 435), and the LAMP-1 N- and C-terminal signal sequences are (SEQ ID NO: 372), and each vector expresses the GM-CSF Gag-Nef and GM-CSF Pol fusion protein sequences (SEQ ID NO: 353 + SEQ ID NO: 363), respectively. The fusion protein sequences are provided in Table J. The A*0201 sequence in the vector contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is cloned into the Ad5 / 35 vector as a bead-and-beads configuration. Figure 35C shows the extent of IFN-γ responses to the A*0201 peptide pool from prime-vaccinated animals. The Y-axis represents the extent of IFN-γ responses to A*0201 peptide pool stimulation as the number of spot-forming cells (SFC) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming. Figure 35D shows the extent of IFN-γ responses to Gag, Nef, Pol-1, and Pol-2 peptide pools in GM-CSF-Gag / Nef+GM-CSF-Pol-vaccinated animals. The vector sequence contains not only A*0201 epitope-specific sequences but also overall conserved sequences. The Y-axis represents the extent of IFN-γ responses in GM-CSF-Gag / Nef+GM-CSCF-Pol vaccine-primed animals, and the X-axis represents the specific peptide pool used for stimulation.Each bar represents stimulation with a peptide pool from Gag p24, Gag p17, Nef, Pol-1 (protease / RT), and Pol-2 (integrase) responses. Responses are expressed as spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. Statistical significance between groups was determined using the nonparametric Mann-Whitney test. *P ≤ 0.05, **P ≤ 0.001. [Figure 35C]Figure 35A shows the immunogenicity of Ad5 / 35 vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 35A depicts the immunization and sampling schedule showing time points for prime vaccination and assessment of responses by IFN-γ ELISpot. Figure 35B shows the immunogen construct used for vaccination, in which the A*0201 sequence without the signal sequence is (SEQ ID NOs: 367, 431), the GM-CSF signal sequence is (SEQ ID NOs: 368, 432), the tPA signal sequence is (SEQ ID NOs: 369, 433), the MCP-3 signal sequence is (SEQ ID NOs: 370, 434), the β-catenin signal sequence is (SEQ ID NOs: 371, 435), and the LAMP-1 N- and C-terminal signal sequences are (SEQ ID NO: 372), and each vector expresses the GM-CSF Gag-Nef and GM-CSF Pol fusion protein sequences (SEQ ID NO: 353 + SEQ ID NO: 363), respectively. The fusion protein sequences are provided in Table J. The A*0201 sequence in the vector contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is cloned into the Ad5 / 35 vector as a bead-and-beads configuration. Figure 35C shows the extent of IFN-γ responses to the A*0201 peptide pool from prime-vaccinated animals. The Y-axis represents the extent of IFN-γ responses to A*0201 peptide pool stimulation as the number of spot-forming cells (SFC) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming. Figure 35D shows the extent of IFN-γ responses to Gag, Nef, Pol-1, and Pol-2 peptide pools in GM-CSF-Gag / Nef+GM-CSF-Pol-vaccinated animals. The vector sequence contains not only A*0201 epitope-specific sequences but also overall conserved sequences. The Y-axis represents the extent of IFN-γ responses in GM-CSF-Gag / Nef+GM-CSCF-Pol vaccine-primed animals, and the X-axis represents the specific peptide pool used for stimulation.Each bar represents stimulation with a peptide pool from Gag p24, Gag p17, Nef, Pol-1 (protease / RT), and Pol-2 (integrase) responses. Responses are expressed as spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. Statistical significance between groups was determined using the nonparametric Mann-Whitney test. *P ≤ 0.05, **P ≤ 0.001. [Figure 35D]Figure 35A shows the immunogenicity of Ad5 / 35 vectors containing conserved HIV sequences with and without leader sequences in A*0201-C57 / BL6 transgenic mice. Figure 35A depicts the immunization and sampling schedule showing time points for prime vaccination and assessment of responses by IFN-γ ELISpot. Figure 35B shows the immunogen construct used for vaccination, in which the A*0201 sequence without the signal sequence is (SEQ ID NOs: 367, 431), the GM-CSF signal sequence is (SEQ ID NOs: 368, 432), the tPA signal sequence is (SEQ ID NOs: 369, 433), the MCP-3 signal sequence is (SEQ ID NOs: 370, 434), the β-catenin signal sequence is (SEQ ID NOs: 371, 435), and the LAMP-1 N- and C-terminal signal sequences are (SEQ ID NO: 372), and each vector expresses the GM-CSF Gag-Nef and GM-CSF Pol fusion protein sequences (SEQ ID NO: 353 + SEQ ID NO: 363), respectively. The fusion protein sequences are provided in Table J. The A*0201 sequence in the vector contains a specific epitope sequence derived from a conserved HIV sequence for the A*0201 allele and is cloned into the Ad5 / 35 vector as a bead-and-beads configuration. Figure 35C shows the extent of IFN-γ responses to the A*0201 peptide pool from prime-vaccinated animals. The Y-axis represents the extent of IFN-γ responses to A*0201 peptide pool stimulation as the number of spot-forming cells (SFC) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. The X-axis indicates the individual vaccine constructs used for in vivo priming. Figure 35D shows the extent of IFN-γ responses to Gag, Nef, Pol-1, and Pol-2 peptide pools in GM-CSF-Gag / Nef+GM-CSF-Pol-vaccinated animals. The vector sequence contains not only A*0201 epitope-specific sequences but also overall conserved sequences. The Y-axis represents the extent of IFN-γ responses in GM-CSF-Gag / Nef+GM-CSCF-Pol vaccine-primed animals, and the X-axis represents the specific peptide pool used for stimulation.Each bar represents stimulation with a peptide pool from Gag p24, Gag p17, Nef, Pol-1 (protease / RT), and Pol-2 (integrase) responses. Responses are expressed as spot-forming units (SFU) per 10 splenocytes. Peptide-specific values ​​were obtained by subtracting the no-peptide stimulation control to exclude nonspecific responses. Statistical significance between groups was determined using the nonparametric Mann-Whitney test. *P ≤ 0.05, **P ≤ 0.001.

[0328] [Figure 36A]Various prime and prime-boost regimens using arenavirus LCMV vectors and Pichinde (PICV) arenavirus vectors are shown. (A) C57Bl / 6 mice were immunized with a single prime with a trisegmented, replicating-attenuated LCMV (TT1) vector, a trisegmented, replicating-attenuated PICV (TT2) vector, a replication-defective PICV (VV2) vector, and an adenovirus vector expressing SIVsme543 gp140. (B) Homologous and heterologous prime-boost regimens were performed with a trisegmented, replicating-attenuated LCMV (TT1) vector or a trisegmented, replicating-attenuated PICV (TT2) vector expressing Gag, Pol-1 / Pol-2, and Env (gp140). (C) Comparison of heterologous prime-boost strategies: first, priming with a trisegmented, replicative-attenuated PICV (TT2) vector or a replication-defective PICV (VV2) vector expressing SIV antigens, followed by boosting with a replication-defective LCMV (VV1) and a trisegmented, replicative-attenuated LCMV (TT1) vector. Mice received a single immunization at each time point consisting of three (trisegmented, replicative-attenuated) or four (replication-defective) arenavirus vectors mixed at a 1:1:1:1 ratio. (D) Immunization of rhesus macaques with a replicative-attenuated arenavirus vector expressing SIVsme543 Gag as the homologous LCMV or PICV and heterologous PICV prime and LCMV boost. Four intravenous immunizations were administered on days 1, 29, 85, and 113. (E) Summary of long-term follow-up immune responses by IFN-γ ELISpot in rhesus macaques immunized with a replicatively attenuated arenavirus vector expressing SIVsme543 Gag as a homologous LCMV or PICV and heterologous PICV prime and LCMV boost. The response observed in (E) is an extension of the response observed in (D). [Figure 36B]Various prime and prime-boost regimens using arenavirus LCMV vectors and Pichinde (PICV) arenavirus vectors are shown. (A) C57Bl / 6 mice were immunized with a single prime with a trisegmented, replicating-attenuated LCMV (TT1) vector, a trisegmented, replicating-attenuated PICV (TT2) vector, a replication-defective PICV (VV2) vector, and an adenovirus vector expressing SIVsme543 gp140. (B) Homologous and heterologous prime-boost regimens were performed with a trisegmented, replicating-attenuated LCMV (TT1) vector or a trisegmented, replicating-attenuated PICV (TT2) vector expressing Gag, Pol-1 / Pol-2, and Env (gp140). (C) Comparison of heterologous prime-boost strategies: first, priming with a trisegmented, replicative-attenuated PICV (TT2) vector or a replication-defective PICV (VV2) vector expressing SIV antigens, followed by boosting with a replication-defective LCMV (VV1) and a trisegmented, replicative-attenuated LCMV (TT1) vector. Mice received a single immunization at each time point consisting of three (trisegmented, replicative-attenuated) or four (replication-defective) arenavirus vectors mixed at a 1:1:1:1 ratio. (D) Immunization of rhesus macaques with a replicative-attenuated arenavirus vector expressing SIVsme543 Gag as the homologous LCMV or PICV and heterologous PICV prime and LCMV boost. Four intravenous immunizations were administered on days 1, 29, 85, and 113. (E) Summary of long-term follow-up immune responses by IFN-γ ELISpot in rhesus macaques immunized with a replicatively attenuated arenavirus vector expressing SIVsme543 Gag as a homologous LCMV or PICV and heterologous PICV prime and LCMV boost. The response observed in (E) is an extension of the response observed in (D). [Figure 36C]Various prime and prime-boost regimens using arenavirus LCMV vectors and Pichinde (PICV) arenavirus vectors are shown. (A) C57Bl / 6 mice were immunized with a single prime with a trisegmented, replicating-attenuated LCMV (TT1) vector, a trisegmented, replicating-attenuated PICV (TT2) vector, a replication-defective PICV (VV2) vector, and an adenovirus vector expressing SIVsme543 gp140. (B) Homologous and heterologous prime-boost regimens were performed with a trisegmented, replicating-attenuated LCMV (TT1) vector or a trisegmented, replicating-attenuated PICV (TT2) vector expressing Gag, Pol-1 / Pol-2, and Env (gp140). (C) Comparison of heterologous prime-boost strategies: first, priming with a trisegmented, replicative-attenuated PICV (TT2) vector or a replication-defective PICV (VV2) vector expressing SIV antigens, followed by boosting with a replication-defective LCMV (VV1) and a trisegmented, replicative-attenuated LCMV (TT1) vector. Mice received a single immunization at each time point consisting of three (trisegmented, replicative-attenuated) or four (replication-defective) arenavirus vectors mixed at a 1:1:1:1 ratio. (D) Immunization of rhesus macaques with a replicative-attenuated arenavirus vector expressing SIVsme543 Gag as the homologous LCMV or PICV and heterologous PICV prime and LCMV boost. Four intravenous immunizations were administered on days 1, 29, 85, and 113. (E) Summary of long-term follow-up immune responses by IFN-γ ELISpot in rhesus macaques immunized with a replicatively attenuated arenavirus vector expressing SIVsme543 Gag as a homologous LCMV or PICV and heterologous PICV prime and LCMV boost. The response observed in (E) is an extension of the response observed in (D). [Figure 36D]Various prime and prime-boost regimens using arenavirus LCMV vectors and Pichinde (PICV) arenavirus vectors are shown. (A) C57Bl / 6 mice were immunized with a single prime with a trisegmented, replicating-attenuated LCMV (TT1) vector, a trisegmented, replicating-attenuated PICV (TT2) vector, a replication-defective PICV (VV2) vector, and an adenovirus vector expressing SIVsme543 gp140. (B) Homologous and heterologous prime-boost regimens were performed with a trisegmented, replicating-attenuated LCMV (TT1) vector or a trisegmented, replicating-attenuated PICV (TT2) vector expressing Gag, Pol-1 / Pol-2, and Env (gp140). (C) Comparison of heterologous prime-boost strategies: first, priming with a trisegmented, replicative-attenuated PICV (TT2) vector or a replication-defective PICV (VV2) vector expressing SIV antigens, followed by boosting with a replication-defective LCMV (VV1) and a trisegmented, replicative-attenuated LCMV (TT1) vector. Mice received a single immunization at each time point consisting of three (trisegmented, replicative-attenuated) or four (replication-defective) arenavirus vectors mixed at a 1:1:1:1 ratio. (D) Immunization of rhesus macaques with a replicative-attenuated arenavirus vector expressing SIVsme543 Gag as the homologous LCMV or PICV and heterologous PICV prime and LCMV boost. Four intravenous immunizations were administered on days 1, 29, 85, and 113. (E) Summary of long-term follow-up immune responses by IFN-γ ELISpot in rhesus macaques immunized with a replicatively attenuated arenavirus vector expressing SIVsme543 Gag as a homologous LCMV or PICV and heterologous PICV prime and LCMV boost. The response observed in (E) is an extension of the response observed in (D). [Figure 36E]Various prime and prime-boost regimens using arenavirus LCMV vectors and Pichinde (PICV) arenavirus vectors are shown. (A) C57Bl / 6 mice were immunized with a single prime with a trisegmented, replicating-attenuated LCMV (TT1) vector, a trisegmented, replicating-attenuated PICV (TT2) vector, a replication-defective PICV (VV2) vector, and an adenovirus vector expressing SIVsme543 gp140. (B) Homologous and heterologous prime-boost regimens were performed with a trisegmented, replicating-attenuated LCMV (TT1) vector or a trisegmented, replicating-attenuated PICV (TT2) vector expressing Gag, Pol-1 / Pol-2, and Env (gp140). (C) Comparison of heterologous prime-boost strategies: first, priming with a trisegmented, replicative-attenuated PICV (TT2) vector or a replication-defective PICV (VV2) vector expressing SIV antigens, followed by boosting with a replication-defective LCMV (VV1) and a trisegmented, replicative-attenuated LCMV (TT1) vector. Mice received a single immunization at each time point consisting of three (trisegmented, replicative-attenuated) or four (replication-defective) arenavirus vectors mixed at a 1:1:1:1 ratio. (D) Immunization of rhesus macaques with a replicative-attenuated arenavirus vector expressing SIVsme543 Gag as the homologous LCMV or PICV and heterologous PICV prime and LCMV boost. Four intravenous immunizations were administered on days 1, 29, 85, and 113. (E) Summary of long-term follow-up immune responses by IFN-γ ELISpot in rhesus macaques immunized with a replicatively attenuated arenavirus vector expressing SIVsme543 Gag as a homologous LCMV or PICV and heterologous PICV prime and LCMV boost. The response observed in (E) is an extension of the response observed in (D).

[0329] [Figure 37A](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. [Figure 37B](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. [Figure 37C](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. [Figure 37D](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. [Figure 37E](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. [Figure 37F](A) Established protocol for moDC-T cell priming assays using a 384-well ELISPOT assay, followed by individual epitopes. (B) Gender, viral load, and HLA diversity profiles currently available for the 10 viremic HIV-1 patient donors who completed this analysis are shown. (C) Viral vector sequences in the absence of signal sequences are shown to assess whether vaccination enhances the breadth of responses assessed in (D). (D) Comparison of the breadth of responses (number of independent epitopes) induced after priming with moDCs transduced with Ad5 / 35 vectors expressing conserved regions within Gag-Nef and Pol-Env. (E) Characterization of the breadth of immune responses targeting different HIV-1 antigens with empty vectors (prevaccines) and conserved region vaccines with different signal sequences (SEQ ID NOs: 353, 363, 354, 355, 356, 429, and 357). (F) Breadth of response, defined as the number of de novo peptide pools (excluding pre-existing baseline responses), and extent of response assessed by IFN-γ ELISpot assay on day 10 after coculture of PBMCs with vaccine vector-transduced autologous moDCs expressing conserved domain constructs with different signal sequences. Each point represents one donor. Means and SDs are shown. DETAILED DESCRIPTION OF THE INVENTION

[0330] 1. Introduction Provided herein are fusion polypeptides comprising multiple polypeptide or peptide segments, as well as related compositions, including immunogenic and pharmaceutical compositions, and methods for making the fusion polypeptides and using them to 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. Polynucleotides encoding the fusion polypeptides described herein, as well as vectors comprising the same, are also provided.

[0331] Provided herein are fusion polypeptides designed to induce antiviral immune responses. The vaccine constructs described herein were designed to provide mathematically determined improved coverage of predicted T cell epitopes ("PTEs") using the most highly conserved predicted epitopes within a source set of viral proteome sequences. Fusion polypeptides encoded by one, two, three, or four of the HIV-1 Gag, Pol, Env, and Nef genes were used as a paradigm for how to design antiviral immunogens. The method described herein preserves positional information for PTEs within the source set of sequences and constructs a bivalent set of sequences to improve coverage of conserved PTEs. The result is an initial bivalent vaccine construct that advantageously improves or increases highly conserved PTEs, which, due to both the preserved positional information, are likely to closely resemble conserved epitopes in naturally occurring sequences in proteins expressed by viral species within patient populations and individual patients. Furthermore, using only highly conserved PTE sequences between patients and among HIV-1 strains within a patient reduces the likelihood of escape mutants, as highly conserved sequences are more likely to contribute to viral structure and function.

[0332] Additionally, a computational approach for designing antiviral vaccine immunogens for highly variable viruses such as HIV-1 is provided. The antiviral vaccine immunogen design method incorporates deep sequencing data from individual patient samples with variable sequences, and sequence diversity is analyzed in the context of host HLA diversity to develop antiviral vaccines for therapeutic and prophylactic use. Antiviral immunogens can be designed to provide coverage at the individual level for groups of individuals with a defined set of HLA alleles, or for broad population coverage. In the vaccine immunogen design method described herein, the inventors use bulk population sequences (e.g., from public and internally developed databases) to define a computational approach for targeting conserved regions within vaccine sequences. Furthermore, deep sequencing data from individual patients is used to define the sequence variability of each potential T cell epitope within the conserved regions. Regions that may serve as actual epitopes are further identified based on the likelihood of presentation by an individual host set of HLA alleles. We developed a deep learning model that models peptide binding per allele using host HLA binding probabilities defined by publicly available and internally developed databases. This can be combined with in silico, published, and / or experimental in vitro T cell priming data to define the potential impact of antigen variants in modulating TCR recognition or identify peptides as escape variants. These data are used to design a set of peptide immunogens containing epitopes and related epitope variants. Epitope sequences are linked or connected sequentially into a single fusion polypeptide, either directly fused or linked via linker sequences. Peptide segments are linked in a computationally determined sequential order from N- to C-terminus, which can mimic human autoantigens and reduce or eliminate the creation of junctional epitopes that may have undesirable effects (e.g., inducing autoimmune or tolerogenic responses).

[0333] Unlike similar graph-based approaches to vaccine design, the approach described herein constructs connected PTE segments using only adjacent PTEs that also flank native sequences. Furthermore, the method first constructs bivalent constructs consisting of two matching polypeptides to improve or increase coverage at each PTE position within the viral proteome. The bivalent construct itself can be used as a vaccine, as in the constructs described in Examples 1 and 2 below, or it can serve as the basis for further constructs (e.g., the HLA-restricted construct described in Example 3 or the personalized constructs described in Examples 4 and 5 below). Bivalent constructs designed by analysis of population-based sequences (e.g., inter-patient diversity) can serve as templates to identify population-based conserved sequences that may contribute to viral structure and function and define conservation within patient sequences; this information can be applied to construct personalized vaccine constructs.

[0334] The methods described herein can begin with identifying conserved region bivalent sequences using a process referred to herein as "conservation analysis" or "conservation algorithm." The methods can further include constructing a bivalent vaccine construct with maximal epitope coverage while retaining PTE position information from the native sequence using a process referred to herein as "conservation walking algorithm" or "CWA."

[0335] Therapeutic vaccines based on the induction of T cell responses have been easily evaluated and shown to be effective in the context of cancer vaccines. These vaccines are typically epitope-based and can be tailored to an individual's HLA alleles as well as their specific tumor antigens (see, e.g., Tran, et al., Science, (2014) 344(6184):641-5). We have developed vaccine sequences that can target subpopulations based on the population prevalence of specific HLA alleles, or can be tailored specifically to an individual's range of HLA alleles. 2. Fusion polypeptides useful for enhancing immune responses against human immunodeficiency virus-1 (HIV-1)

[0336] Provided herein are fusion polypeptides comprising multiple polypeptide or peptide segments encoded by one or more HIV-1 genes. A "segment" of a fusion polypeptide described herein is a contiguous 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 polypeptides described herein are "fusion" polypeptides in the sense that they are assembled from connected or linked polypeptide or peptide segments of two or more HIV-1 proteins. With respect to the HIV-1 protein reference sequence, the polypeptide or peptide segments can correspond to discontinuous sequences of the same HIV-1 protein or different HIV-1 proteins. Generally, fusion polypeptides are non-naturally occurring and can be synthetically or recombinantly produced. a. polypeptide segment

[0337] With respect to the HIV-1 genes encoding the polypeptide segments used to construct the fusion polypeptides described herein, in various embodiments, the fusion polypeptide comprises multiple polypeptide segments of one or more human immunodeficiency virus-1 (HIV-1) proteins encoded by one or more (e.g., two or more, three or more, four or more) HIV-1 genes (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 multiple polypeptide segments consist exclusively of polypeptide segments encoded by the HIV-1 genes Env, Gag, Nef, and Pol, and do not include, for example, polypeptide segments encoded by the HIV-1 Tat, Rev, Vif, Vpr, and / or Vpu genes. In some embodiments, the plurality of polypeptide segments consists solely of polypeptide segments encoded by the HIV-1 genes Gag, Nef, and Pol, e.g., 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 solely of polypeptide segments encoded by the HIV-1 genes Gag and Nef, e.g., 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 solely of polypeptide segments encoded by the HIV-1 genes Pol and Nef, e.g., 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 solely of polypeptide segments encoded by the HIV-1 genes Pol and Env, e.g., 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 solely of polypeptide segments encoded by HIV-1 Pol genes, e.g., does not include polypeptide segments encoded by HIV-1 Env, Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes. In various embodiments, the plurality of polypeptide segments consists solely of polypeptide segments encoded by HIV-1 Pol genes, e.g., does not include polypeptide segments encoded by HIV-1 Env, Gag, Nef, Tat, Rev, Vif, Vpr, and / or Vpu genes. It does not contain segments encoded by one, two, three, or four of the Tat, Rev, Vif, Vpr, and / or Vpu genes.

[0338] With respect to the number of polypeptide segments assembled, connected, linked, or concatenated into a single fusion polypeptide, in various embodiments, the fusion polypeptide is comprised of at least 5 and up to 40 polypeptide segments, e.g., 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. If desired, the polypeptide segments can be arranged in the same order or in a different order than in the naturally occurring protein.

[0339] With respect to regions of polypeptides encoded by HIV-1 genes selected as polypeptide segments comprising the fusion polypeptide, in various embodiments, the polypeptide segments are derived from conserved regions of a population of viral proteome sequences. In some embodiments, the conserved regions are greater than 80%, e.g., greater than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% conserved among HIV-1 strains, e.g., as determined in inter- and / or intra-patient populations. 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 9 amino acid long or 9-mer segment) as the most prevalent at a given amino acid segment or subsequence position, where the amino acid segment or subsequence position is determined relative to a reference sequence (e.g., an HIV-1 HXB2 polypeptide sequence (e.g., SEQ ID NOS: 403-406)). In various embodiments, the conserved region is conserved among one or more 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., between HIV-1 Group M, Clade B, and its recombinant forms, e.g., CRF01_AE. In some embodiments, the plurality of polypeptide segments comprises at least two polypeptide segments, 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 selected from SEQ ID NOs: 1-344, e.g., the polypeptide segments identified in Table B.In some embodiments, the plurality of polypeptide segments comprises the sequences of 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, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 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, 99, 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, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 3 and 322, 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 C.The start and end positions are provided herein as SEQ ID NOS: 403-406 and identified in Table A, relative to the HIV-1 HXB2 reference polypeptide, GenBank accession number 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 2-11] [Table 2-12] [Table 2-13] [Table 3-1]

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

[0340] With regard to the range of lengths of the individual polypeptide 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, e.g., at least 8 amino acids in length and up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. In various embodiments, each polypeptide segment is at least 8 amino acids in length and up to about 35 amino acids in length, e.g., at least 8 amino acids in length and up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, or 35 amino acids in length. In various embodiments, each polypeptide segment is at least 15 amino acids in length and up to about 30 amino acids in length, e.g., at least 15 amino acids in length and up to 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0341] With respect to the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full-length fusion polypeptide is at least about 350 amino acids and up to about 1000 amino acids, e.g., 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. With respect to the length of the full-length fusion polypeptide, in various embodiments, in some embodiments, the full-length fusion polypeptide comprises at least about 350 amino acids and up to about 800 amino acids, e.g., 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 total 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.

[0342] Generally, fusion polypeptides are immunogenic in that they are capable of inducing an immune response in humans, e.g., against HIV-1. In some embodiments, the fusion polypeptides, optionally in combination with, e.g., one or more additional therapeutic agents as described herein, can induce a protective or therapeutically effective immune response against HIV-1 in humans, e.g., prevent HIV-1 infection in uninfected individuals, or, in a therapeutic setting, induce an immune response sufficient to induce immune-mediated control of or eradicate HIV-1 in infected individuals. The immunogenicity of the fusion polypeptides can be assessed and demonstrated in in vitro and in vivo assays, as described herein. For example, the immunogenicity of the fusion polypeptides can be demonstrated by in vitro assays, including CD4+ and / or CD8+ T cell activation (e.g., 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 a fusion polypeptide can also be demonstrated in an in vivo animal model, for example, by administering to mice transgenic for one or more human HLA molecules (available from Jackson Laboratories or Taconic) or non-human primates and assessing CD4+ and / or CD8+ T cell activation (including, for example, serum cytokine levels) or proliferation. In various embodiments, one, two, three, or more of each polypeptide segment comprises or consists of one or more predicted T cell epitopes, e.g., determined computationally or experimentally. In some embodiments, the fusion polypeptide comprises 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), e.g., within a single subject or between multiple subjects. In some embodiments, the fusion polypeptide comprises at least one polypeptide segment that binds to or is ...).*

[0201] Comprises one or more polypeptide segments that bind to or are presented by HLA class I molecules. In some embodiments, a fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more 8-mer, 9-mer, and / or 10-mer polypeptide 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), e.g., within a single subject. In some embodiments, the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more polypeptide segments, each 15-30 amino acids in length, that are processed intracellularly and presented by one or more human HLA class I and / or class II alleles (e.g., 1, 2, 3, 4, 5, or 6 alleles), e.g., within a single subject. Concatenated Polypeptide Segments

[0343] Optionally, one or more of the polypeptide segments may be directly abutted or fused to adjacent segments, or may be bonded, connected, or linked to adjacent segments by one or more peptide linkers. In various embodiments, the 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, for example, within the linker or within the full-length fusion polypeptide. Exemplary fusion protein linkers that can be used in the fusion polypeptides of the present 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, the polyalanine linker comprises or consists of two or three consecutive alanine residues, e.g., AA, AAA (SEQ ID NO: 378), AAY (SEQ ID NO: 379), or AAX (wherein X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y) (SEQ ID NO: 380). In some embodiments, a polyglycine linker is used, e.g., GGS (SEQ ID NO: 419), GSG (SEQ ID NO: 420), or GGGS (SEQ ID NO: 421).

[0344] 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 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. 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, a furin recognition / cleavage sequence (e.g., REKR (SEQ ID NO: 382), RRKR (SEQ ID NO: 383), RAKR (SEQ ID NO: 381)) is combined or fused in a single linker with a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A)). See, e.g., Chng, et al., mAbs (2015) 7:2, 403-412. In various embodiments, the 2A cleavable linker is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 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%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188, 189%, 1 8%, or at least 99% identical to, and comprising or consisting 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 may be located at either the N- or C-terminus of the 2A linker.In some embodiments, the cleavable linker comprises or consists of a furin recognition / cleavage site selected from the group consisting of RAKR (SEQ ID NO: 381), REKR (SEQ ID NO: 382), and RRKR (SEQ ID NO: 383). REKR (SEQ ID NO: 382) is a cleavable linker naturally occurring in HIV and SIV envelope glycoprotein precursors (Bahbouhi, et al., Biochem. J. (2002) 366, 863-872). In some embodiments, one or more Nef linking sequences comprise or consist 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 comprise or consist 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 D. [Table 4] Polypeptide segments encoded by the HIV-1 Gag gene

[0345] In various embodiments, the fusion polypeptide comprises one or more segments, or fragments or subsequences thereof, of one or more viral proteins encoded by the HIV-1 Gag gene. 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-terminus). In some embodiments, the one or more viral proteins encoded by the HIV-1 Gag gene do not include any p6 components.In some embodiments, the plurality of polypeptide segments comprises 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 to 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 to 79 and 92 to 124, SEQ ID NOs: 70 to 71, 76 to 77 and 94 to 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 at least two polypeptide segments, 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 segments, comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 78, 137.

[0346] In some embodiments, the fusion polypeptide comprises at least two polypeptide segments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more segments, comprising or consisting of HIV-1 Gag amino acid sequences corresponding to 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, wherein the amino acid positions are relative to SEQ ID NO:404. In certain embodiments, the fusion polypeptide does not comprise one, two, three, four, five, or more polypeptide segments comprising or consisting of an HIV-1 Gag amino acid sequence or subsequence thereof corresponding to amino acid residue positions selected from 1-30, 54-127, 138-146, 370-428, and 445-500, where the amino acid positions are based on SEQ ID NO:404. In some embodiments, the plurality of polypeptide segments does not include, and the fusion proteins described herein do not include, one, two, three, four, five, or more polypeptide segments that comprise or consist of the HIV-1 Gag amino acid sequence of any one of SEQ ID NOs: 444-448, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 444-448, or a subsequence thereof (Table K). Exemplary polypeptide segments encoded by the Gag gene and incorporated into the fusion polypeptides described herein (e.g., determined to be derived from conserved regions and associated with human HLA A *0201 and / or known to be immunogenic) are shown aligned to the HIV-1 HXB2 Gag reference polypeptide in Figure 18. As used herein, the numbering of a given amino acid polymer or nucleic acid polymer "corresponds to," "corresponds to," or "relative to" the numbering of a selected or reference amino acid polymer or nucleic acid polymer when the position of any given polymer component (e.g., amino acid, nucleotide, also commonly referred to as "residue") is designated by reference to the same or equivalent position in the selected amino acid or nucleic acid polymer (e.g., based on an optimal alignment or consensus sequence), rather than by the actual numerical position of the component in the given polymer. Polypeptide segments encoded by the HIV-1 Nef gene

[0347] 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 multiple polypeptide segments are selected from the group consisting of 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; 50, 155, 156, 157, 158, 159, 160, 166, 167, 168, 169, 170 and 171, SEQ ID NOs: 149 to 152, SEQ ID NOs: 151 to 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 NOs:153 and 165, and SEQ ID NO:153.

[0348] In some embodiments, the fusion polypeptide comprises at least two polypeptide segments, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more segments, comprising or consisting of HIV-1 Nef amino acid sequences corresponding to 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, where the amino acid positions are based on SEQ ID NO:405. In certain embodiments, the fusion polypeptide does not include, or the fusion proteins described herein do not include, one, two, three, or more polypeptide segments comprising or consisting of an HIV-1 Nef amino acid sequence, or a subsequence thereof, corresponding to amino acid residue positions selected from among 1-63, 103-116, and 155-206, where the amino acid positions are based on SEQ ID NO: 405. In some embodiments, the plurality of polypeptide segments does not include, or the fusion proteins described herein do not include, one, two, three, or more polypeptide segments comprising or consisting of an HIV-1 Nef amino acid sequence of any one of SEQ ID NOs: 449-451, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 449-451, or a subsequence thereof (Table K). Exemplary polypeptide segments (e.g., determined to be derived from conserved regions and associated with human HLA A) encoded by the HIV-1 Nef gene and incorporated into the fusion polypeptides described herein are: * 0201 and / or known to be immunogenic) are shown aligned to the HIV-1 HXB2 Nef reference polypeptide in FIG. Fusion polypeptides having polypeptide segments encoded by HIV-1 Gag and Nef genes

[0349] 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, e.g., excluding 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 ... 52 and 162. Polypeptide segments that may be included in fusion polypeptides having polypeptide segments encoded by the HIV-1 Gag and Nef genes are listed in Table E. [Table 5-1] [Table 5-2] [Table 5-3]

[0350] In some embodiments, the fusion polypeptide comprises or consists of, in N-terminal to C-terminal order, the polypeptide segments of SEQ ID NOs: 70, 76, 94, 151 and 161, or SEQ ID NOs: 71, 77, 95, 152 and 162, optionally joined or connected by one or more linkers.

[0351] 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.

[0352] Modifications may be made in the structure of the fusion polypeptides described herein and the polynucleotides encoding such fusion polypeptides to obtain functional molecules that encode variant or derivative polypeptides that still have desired (e.g., immunogenic) properties. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or further improved variant or portion of a fusion polypeptide described herein, one skilled in the art would typically change one or more of the codons in the encoding DNA sequence.

[0353] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind to other polypeptides (e.g., antigens) or cells. Because it is the binding ability and properties of a protein that define its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence, and of course, in the underlying DNA coding sequence, to nevertheless obtain a protein with similar properties. Thus, it is contemplated that various changes can be made in the polypeptide sequences of the disclosed fusion polypeptides, or the corresponding DNA sequences encoding such fusion polypeptides, without appreciable loss of their biological utility or activity.

[0354] Often, a polypeptide variant will contain one or more conservative substitutions, a "conservative substitution" being one in which an amino acid is substituted with another amino acid of similar properties, which one skilled in the art of peptide chemistry would expect to leave the secondary structure and hydrogenolytic properties of the polypeptide substantially unchanged.

[0355] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, where a sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned.

[0356] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) with default parameters. This program embodies multiple alignment schemes, as described in the following references: Dayhoff, MO (1978) A model of evolutionary change in protein-Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358, Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, CA, Higgins, DGand ​​Sharp, PM (1989) CABIOS 5:151-153, Myers, EWand Muller W. (1988) CABIOS 4:11-17, Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425, Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA, Wilbur, WJand. Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0357] Alternatively, optimal alignment of sequences for comparison may be performed 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, or by the 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.

[0358] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0359] In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). Extension of word hits in each direction is halted when the cumulative alignment score falls off by an amount X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when 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 as defaults a word length (W) of 11 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, a word length (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0360] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls off by an amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0361] In one approach, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5-15%, or 10-12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur 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., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0362] As used herein, a "polypeptide variant" is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be synthetically produced, for example, by modifying one or more of the above polypeptide sequences described herein and assessing 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" can also refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations. Polypeptide segments encoded by the HIV-1 Env gene

[0363] In some embodiments, the fusion polypeptide comprises one or more segments of one or more viral proteins encoded by the HIV-1 Env gene, hi certain embodiments, the one or more viral proteins encoded by the HIV-1 Env gene are selected from gp120 and gp41.

[0364] In various embodiments, the fusion polypeptide is selected from the group consisting of SEQ ID NOS: 1-67 and 338, SEQ ID NOS: 2, 3, 8, 9, 13, 1...

Claims

[Claim 1] The invention as depicted in the drawings.

Images