WO2018195447A1 - Recombinant mva-based hiv immunogens and uses thereof - Google Patents

Abstract

Compositions and methods suitable for use in inducing anti-HIV-1 antibodies are provided. Immunogenic compositions comprise recombinant nucleic acids, recombinant modified vaccinia Ankara (MVA) based vectors and recombinant Env proteins to induce antibody lineages capable of generating cross-reactive neutralizing antibodies and to direct the evolution of their breadth of coverage. The invention also relates to methods of inducing an immune response to HIV using such compositions and more specifically to inducing B cell lineages producing broadly neutralizing anti-HIV-1 antibodies.

Description

RECOMBINANT MVA-BASED HIV IMMUNOGENS AND USES THEREOF

[1] This invention was made with government support under grant R43AI120887 awarded by the Department of Health and Human Services, National Institutes of Health and support under grant UM1 -All 00645 awarded by NIAID, NIH. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[2] This application claims the benefit of U.S. provisional patent application 62/487,939 filed April 20, 2017 the contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

[3] The present invention relates to a composition suitable for use in inducing anti -HIV-1 antibodies, and, in particular, to immunogenic compositions comprising recombinant nucleic acid vectors, recombinant modified vaccinia Ankara (MVA) based vectors and recombinant envelope (Env) proteins to induce antibody lineages capable of generating cross-reactive neutralizing antibodies and to direct the evolution of their breadth of coverage. The invention also relates to methods of inducing and immune response to HIV using such compositions and more specifically to induce B cell lineages producing broadly neutralizing anti -HIV-1 antibodies.

BACKGROUND

[4] The development of a safe and effective HIV-1 vaccine is one of the highest priorities of the scientific community working on the HIV-1 epidemic. While anti -retroviral treatment (ART) has dramatically prolonged the lives of HIV-1 infected patients, ART is expensive, not routinely available in many countries and requires rigorous lifelong use.

[5] For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. In the absence of an approved vaccine, antiretroviral drugs are used for prophylactic and therapeutic purposes. Highly active antiretroviral therapy (HAART) is used to treat HIV-1 infection and impede development of AIDS, but HAART fails to adequately target the latently-infected cells that serve as a reservoir for HIV-1. There remains a need for an effective vaccine for prophylactic and therapeutic use. SUMMARY OF THE INVENTION

[6] The present invention addresses the unmet need for a vaccine to prevent and treat HIV. The present invention includes compositions and methods for inducing a neutralizing antibody (nAb) response to HIV. Compositions expressing HIV Env lineage members or natural variants of an HIV T/F Env are described and used in methods of for inducing a neutralizing antibody (nAb) response to HIV.

[7] In a first aspect the invention provides, a recombinant modified vaccinia Ankara (MVA) vector comprising a nucleic acid insert encoding one or more HIV immunogens that elicit an immune response against an HIV virus wherein the insert comprises an HIV gag sequence, and a sequence encoding HIV Env, or lineage-related HIV Env of an HIV T/F Env.

[8] In some embodiments the recombinant MVA vector comprises a nucleic acid insert encoding one or more HIV Env proteins, wherein the one or more HIV Env proteins are (i) identified from an acute or chronic natural infection or (ii) a laboratory-derived mutant of an HIV Env protein identified from an acute or chronic natural infection.

[9] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits an immune response to HIV.

[10] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits a protective immune response to HIV infection.

[11] In one embodiment, the immune response is a T cell response.

[12] In one embodiment, the immune response is a B cell response.

[13] In one embodiment, the immune response is an antibody (Ab) response.

[14] In one embodiment, the immune response is a broadly neutralizing antibody (bnAb) response.

[15] In one embodiment, the HIV immunogens are selected from HIV Gag and T/F HIV Env selected from HIV subtype A, B, C, D, F, G, H, J or K.

[16] In one embodiment, the HIV Env is selected from CH0505 T/F Env, CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week 100.B6 Env.

[17] In one embodiment, the recombinant MVA vector comprises SEQ ID NO: 13 (FIG. 5C - CH0505 T/F gag sequence placed in the MVA vector) and SEQ ID NO: 2 (Fig.4C -CH0505 T/F gpl50 env sequence that is placed in the MVA vector).

[18] In one embodiment, the recombinant MVA vector comprises SEQ ID NO: 13 (FIG. 5C - CH0505 T/F gag sequence placed in the MVA vector) and SEQ ID NO: 4 (FIG. 4E -CH0505 week 53.16 env sequence placed in the MVA vector).

[19] In one embodiment, the recombinant MVA vector comprises SEQ ID NO: 13 (FIG. 5C - CH0505 T/F gag sequence placed in the MVA vector) and SEQ ID NO: 6 (FIG. 4G -CH0505 week 78.33 env sequence that is placed in the MVA vector).

[20] In one embodiment, the recombinant MVA vector comprises SEQ ID NO: 14 (FIG. 5C - CH0505 T/F gag sequence placed in the MVA vector) and SEQ ID NO: 8 (FIG. 41 -CH0505 week 100.B6 env sequence that is placed in the MVA vector).

[21] In one embodiment, the recombinant MVA vector comprises the sequence between positions 1628 and 3838 from plasmid pLW73.

[22] In other embodiments, the recombinant MVA vector comprises the corresponding sequence between positions 1628 and 3838 from plasmids based on pLW73, comprising

CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week 100.B6 Env.

[23] In other embodiments, the recombinant MVA vector comprises the corresponding sequence between positions 1628 and 3838 from plasmids based on pLW73, comprising gpl40 of CH0505 T/F, CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week 100.B6 Env.

[24] In one embodiment, the recombinant MVA vector comprises the sequence between positions 1578 and 3077 from plasmid pLW76.

[25] In one embodiment, the recombinant MVA vector comprises inserts operably linked to a promoter such that cellular expression of the gag and env sequences produce of virus-like particles (VLPs) from the cells of the individual receiving the immunogenic vector compositions described herein.

[26] In another embodiment, the HIV Env is selected from EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env. [27] In another embodiment, the EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[28] In some embodiments the UCA is V_H VH4-4 and VL3-25, VH1-46/1-2, VK3-20, or VH3-49 and Vkl-49 for bnAb to the CD4bs.

[29] In one embodiment, the recombinant MVA vector further comprised a promoter operably linked to the nucleic acid insert such that cellular expression of the HIV gag and HIV env produces of virus-like particles (VLPs).

[30] In a second aspect, the invention provides recombinant deoxyribonucleic acid (DNA) vector comprising an insert comprising consecutive nucleic acids comprising an HIV gag sequence, and a sequence encoding an HIV T/F Env, or lineage-related HIV Env protein of the HIV T/F Env, wherein one or more HIV immunogens that elicit an immune response against an HIV virus.

[31] In one embodiment, the HIV Env is (i) identified from an acute or chronic natural infection, or (ii) a laboratory-derived mutant of an HIV Env protein identified from an acute or chronic natural infection.

[32] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits an immune response to HIV.

[33] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits a protective immune response to HIV infection.

[34] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits a protective immune response to HIV infection.

[35] In one embodiment, the immune response is a T cell response.

[36] In one embodiment, the immune response is a B cell response.

[37] In one embodiment, the immune response is an antibody (Ab) response.

[38] In one embodiment, the immune response is a broadly neutralizing antibody (bnAb) response.

[39] In one embodiment, the immunogens are HIV Gag, and an HIV Env protein selected from CH0505 T/F Env, CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week ΙΟΟ.Βό Εην.

[40] In one embodiment, the immunogens are CH0505 T/F gag, and an HIV Env protein selected from CH0505 T/F Env, CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week 100.B6 Env.

[41] In some embodiments of the recombinant DNA vector, the Env immunogen is SEQ ID NO: 16 (Figure7B -CH0505 T/F-gpl60 placed in the DNA vector).

[42] In some embodiments of the recombinant DNA vector, the Env immunogen is SEQ ID NO: 15 (Figure6B -CH0505 T/F-gpl50 placed in the DNA vector).

[43] In some embodiments of the recombinant DNA vector, the Env immunogen is gpl40 Env CH505 T/F.

[44] In another embodiment, the one or more HIV immunogens are selected from EB354 Gag, and any one of the following Envs: EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env.

[45] In another embodiment, the EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[46] In some embodiments the UCA is VH4-4 and VL3-25, VH1-46/1-2, VK3-20, or VH3-49 and Vkl-49 for bnAb to the CD4bs.

[47] In some embodiments of the recombinant DNA vector or the recombinant MVA vector, the HIV Env immunogen is a gpl60, gpl50 or gpl40 Env.

[48] In some embodiments, the DNA vectors produce Virus-Like Particle (VLP) from the cells of the individual receiving the immunogenic vector compositions described herein.

[49] In a third aspect the invention provides a pharmaceutical composition comprising one or more recombinant MVA vectors described herein, or one or more recombinant DNA vectors described herein, wherein the one or more MVA or DNA vectors comprises an HIV gag sequence, and a sequence encoding HIV Env, and wherein the composition comprises a pharmaceutically acceptable carrier.

[50] In one embodiment, the pharmaceutical composition comprises two or more recombinant MVA or DNA vectors encoding the same or different HIV Env proteins.

[51] In one embodiment, the modified vaccinia Ankara (MVA) vectors express VLPs displaying HIV Env immunogens.

[52] In some embodiments the HIV Env is from transmitted founder viruses and lineages of the T/F virus that are selected in infected cells for the elicitation of bnAb.

[53] In some embodiments the HIV Env protein is identified from an acute or chronic natural infection or a laboratory-derived mutant of an HIV Env protein identified from an acute or chronic natural infection.

[54] In some embodiments the HIV Env is any one of the following CH0505 Envs: T/F, week 53.16, week 78.33, and week 100.B6.

[55] In another embodiment, the one or more HIV immunogens are selected from HIV Gag, and any one of the following Envs: EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env.

[56] In another embodiment, the EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[57] In some embodiments the UCA is VH4-4 and VL3-25, VH1-46/1-2 and VK3-20, or VH3-49 and Vkl-49 germ line sequences for bnAb to the CD4bs.

[58] In some embodiments, the composition comprises a suitable adjuvant.

[59] In various embodiments, the adjuvant is selected from mineral gels such as aluminum hydroxide, aluminum salts (e.g., aluminum phosphate) or calcium salts (e.g., calcium phosphate); MF59, or SAF; adjuvant systems (AS01, AS02, AS03, AS04) (GlaxoSmithKline), complete Freund's adjuvant, incomplete Freund's adjuvant, microbially-derived adjuvants such as cholera toxin (CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72), Bacille Calmette-Guerin (BCG), lipopolysaccharides (LPS), mycobacteria, tetanus toxin, Corynebacterium parvum, DNA CpG motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate adjuvants such as immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres, or saponins (e.g., QS-21); cytokines such as IFN-γ, IL-1, IL-2, IL- 12 or GM-CSF; synthetic adjuvants such as nonionic block copolymers or surfactants, muramyl peptide analogues (e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine [thr-MDP], N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[l'- 2'-dipalmitoyl-s- n-glycero-3-hydroxyphosphoryloxy]-ethylamine), polyphosphazenes, synthetic polynucleotides, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, hydrocarbon emulsions, or keyhole limpet hemocyanins (KLH), CMC (carboxyl methylcellulose), HPMC (hydroxypropyl methylcellulose), glucopyranosyl Lipid adjuvant (GLA), or polylCLC.

[60] In some embodiments the pharmaceutical composition comprises a DNA prime vector to prime an immune response and an MVA boost vector to boost a primed immune response wherein the DNA vector encodes an HIV Env that binds with an UCA for bnAb, and the MVA boost vector comprises a sequence encoding the same Env of the DNA prime vector.

[61] In some embodiments the UCA is V_H4-59 for bnAb to the CD4bs.

[62] In some embodiments the DNA or MVA-expressed VLPs present Env proteins from infected individual CH0505.

[63] In some embodiments of the invention the expressed Env protein immunogen is the gpl60 form of the CH0505 transmitted/founder (T/F) Env.

[64] In some embodiments of the invention the expressed Env protein is a partially truncated Env such as a gpl50, gpl40, gpl20 Env or a mutant CH0505 T/F Env.

[65] In certain embodiments the composition comprises four CH0505 Env proteins for eliciting neutralizing Ab directed to the CD4bs— the T/F Env, the week 53.16 Env, the week 78.33 Env, and the week 100.B6 Env, wherein sequences encoding the Envs are inserted in DNA and/or MVA vectors, wherein in certain embodiments the Envs are produced in the vaccinated person and in some embodiments are displayed on VLPs.

[66] In another embodiment, the one or more HIV immunogens are selected from HIV Gag, and any one of the following Envs: EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env.

[67] In another embodiment, the EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[68] In some embodiments the UCA is VH4-4 and VL3-25, VH1-46/1-2 and VK3-20, or VH3-49 and Vkl-49 for bnAb to the CD4bs. [69] In certain embodiments the composition comprises four EB354 Env proteins for eliciting neutralizing Ab directed to the CD4bs - the T/F Env, the 2010 Env, the 2014 Env, and the 2015 Env, wherein sequences encoding the Envs are inserted in DNA and/or MVA vectors, wherein in certain embodiments the Envs are produced in the vaccinated person and in some embodiments are displayed on VLPs.

[70] In some embodiments of the invention the pharmaceutical composition further comprises (i) a protein boost selected from recombinant gpl20 protein, gpl20deltaN, gpl40 SOSIP trimer, and/or (ii) a recombinant Env binding a germ-line Ig sequence for bnAb.

[71] In one embodiment, the pharmaceutical composition comprises a DNA prime-MVA boost immunogenic composition in which the DNA Ab composition comprises an HIV Env that binds with the UCA to a CD4bs lineage antibody and the boost comprises the same or different Env in an MVA vector. See Liao et al. Nature 496 (2013):469-476 including supplementary materials, and Bonsignori et al. Cell 165 (2016):449-63. doi: 10.1016/j .cell.2016.02.022. Epub 2016 Mar 3, and see also WO2014/042669 for CH0505 immunogens.

[72] In one embodiment, the priming DNA composition comprises a HIV Env protein displayed on VLPs that interacts with UCA for bnAb and the boosting composition comprises the same Env in an MVA, wherein the Env protein is displayed on VLPs.

[73] In one embodiment, the priming DNA composition comprises the CH0505

transmitted/founder (T/F) Env protein that interacts with the V_H4-59 UCA for bnAb for the CD4bs and the boosting MVA composition comprises the same Env.

[74] In one embodiment, the boosting composition can include a gpl20 protein, a

gpl20deltaN protein, or gpl40 SOSIP trimer to enhance responses elicited by the native Envs displayed on VLPs by the DNA and MVA vectors. The gpl20 or gpl40 SOSIP trimer proteins can be delivered at the same time or different times than the DNA or MVA inoculations.

[75] In a fourth aspect, a method is provided to induce an immune response to HIV comprising: administering to a subject an effective amount of the recombinant MVA vector of the invention, or the recombinant MVA vector of the invention, or a combination thereof to prime an immune response, wherein the vectors comprise a nucleic acid insert comprising an HIV gag sequence and a sequence encoding an HIV Env, thereby inducing an immune response.

[76] In various embodiments, the method provides administration regimens comprising administering the DNA and MVA vectors of the invention plus recombinant gpl20,

gpl20deltaN, or gpl40 SOSIP trimer protein subunits in amounts and manners sufficient to induce an immune response.

[77] In one embodiment, the HIV Env protein or mutant HIV Env protein elicits a protective immune response to HIV infection.

[78] In one embodiment, the immune response is a T cell response.

[79] In one embodiment, the immune response is a B cell response.

[80] In one embodiment, the immune response is an antibody (Ab) response.

[81] In one embodiment, the immune response is a broadly neutralizing antibody (bnAb) response.

[82] In some embodiments, the antibodies are CD4bs antibodies.

[83] In some embodiments, the neutralizing antibodies are autologous.

[84] In one embodiment, the compositions described herein comprising one or more vectors and/or protein boosts are administered at intervals of at 3 weeks.

[85] In one embodiment, the compositions described herein comprising one or more vectors and/or protein boosts are administered at intervals of at least 3 weeks.

[86] In various embodiments, the compositions described herein comprising one or more vectors and/or protein boosts are administered at intervals of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks and and greater than 16 weeks.

[87] In some embodiments, the recombinant DNA vector or a pharmaceutical composition comprising the recombinant DNA vector is administered as a prime and the recombinant MVA vector or a pharmaceutical composition comprising the recombinant MVA vector is administered as a boost.

[88] In one embodiment, the method comprises administering a DNA prime-MVA boost immunogenic composition in which the DNA vector encodes an HIV Env that binds with an UCA and the MVA vector boost expresses the same Env. [89] In one embodiment, the nucleic acid insert of both the recombinant DNA vector and the recombinant MVA vector encodes an HIV Env displayed on VLPs that binds with an UCA for bnAb, wherein the HIV Env is the same expressed from both vectors.

[90] In one embodiment, the nucleic acid insert of both the DNA vector and the MVA vector encodes CH0505 T/F Env, and wherein the CH0505 T/F Env is displayed on VLPs and binds with an UCA for bnAb.

[91] In one embodiment, the DNA vector composition comprises the CH0505 T/F Env displayed on VLPs that binds with an UCA for bnAb and the MVA boost comprises a different Env, wherein the different HIV Env is displayed on VLPs.

[92] In one embodiment, the DNA vector and the MVA vector encodes EB354 T/F Env, and wherein the EB354 Env is displayed on VLPs that binds with an UCA for bnAb.

[93] 32. The method of claim 25 wherein the DNA vector composition comprises the EB354 T/F Env displayed on VLPs that binds with an UCA for bnAb and the MVA boost comprises a different Env, wherein the different HIV Env is displayed on VLPs.

[94] In one embodiment, the method for inducing an immune response to an HIV Env that binds an UCA for bnAb to the CD4bs comprises the CH0505 transmitted/founder (T/F) Env on VLPs that interacts with the V_H4-59 UCA for bnAb on precursor B cells and the boosting composition comprises the same Env in an MVA vector, wherein the Env is displayed on VLPs. In some embodiments the boosting composition comprises a different Env in an MVA vector, and/or as a recombinant protein.

[95] In one embodiment, the method for inducing an immune response to an HIV Env that binds an UCA for bnAb to the CD4bs comprises the EB354 transmitted/founder (T/F) Env on VLPs that interacts with the V_H4-4 and V_L3-25, Vl-46/1-2, VK3-20, or VH3-49 and Vkl-49 UCA for bnAb on precursor B cells and the boosting composition comprises the same Env in an MVA vector, wherein the Env is displayed on VLPs. In some embodiments the boosting composition comprises a different Env in an MVA vector, and/or as a recombinant protein.

[96] In one embodiment, the composition further comprises a gpl20 protein, a gpl20deltaN, or gpl40 SOSIP trimer to enhance responses primed by the Envs displayed on VLPs by the DNA and MVA immunogenic compositions.

[97] In some embodiments, the recombinant MVA vector or the pharmaceutical composition comprising the recombinant MVA vector is administered as both a prime and a boost.

[98] In some embodiments, the method comprises administering a heterologous or homologous gpl20 Env protein, gpl20deltaN protein or gpl40 SOSIP trimer protein as a boost.

[99] In one embodiment, the method comprises a) administering DNA vector encoding CH0505 T/F HIV Env as gpl60 or gpl50, b) administering an MVA vector encoding CH0505 T/F HIV Env as gpl50 or gpl40, b) administering a boost with one or more of i) a recombinant gpl20 T/F HIV Env, ii) gpl20deltaN T/F HIV Env or iii) gpl40 SOSIP trimer HIV Env.

[100] In one embodiment, the DNA vector encodes CH505 T/F Env as gpl60.

[101] In one embodiment, the DNA vector encodes CH505 T/F Env as gpl50.

[102] In one embodiment, the MVA vector encodes CH505 T/F Env as gpl50.

[103] In one embodiment, the MVA vector encodes CH505 T/F Env as gpl40.

[104] In one embodiment, the MVA vector is administered at least two times.

[105] In one embodiment, the MVA vector is administered 2-4 times, 2-6 times, or 2-8 times.

[106] In one embodiment, the method comprises: administering DNA comprising CH0505 T/F HIV Env as gpl60, administering an MVA vector comprising CH0505 T/F HIV Env as gpl50, administering a boost with recombinant MVA comprising gpl50 of CH0505 T/F HIV Env, CH0505 week 53.16 HIV Env, CH0505 week 78.33 HIV Env, or CH0505 week 100.B6 HIV Env, administering a boost with a recombinant gpl20, gpl20deltaN or gpl40 SOSIP trimer for CH0505 T/F HIV Env, week 53.16 HIV Env, week 78.33 HIV Env, or week 100.B6 Envs. [107] In one embodiment, the method comprises

[108] administering a DNA vector comprising CH0505 T/F Env as gpl60 or gpl50,

[109] administering an MVA vector comprising CH0505 T/F Env as gpl50 or gpl40,

[110] administering a boost with recombinant MVA comprising gpl50 or gpl40 forms of CH0505 week 53.16 Env, week 78.33 Env, or week 100.B6 Env,

[111] administering a boost with a recombinant Env protein comprising gpl20, gpl20deltaN or gpl40 SOSIP trimer forms of CH0505 T/F Env, week 53.16 Env, week 78.33 Env or week 100.B6 Env.

[112] In some embodiments, the methods comprise administering DNA or MVA encoding the HIV Env. The expressed Env can be gpl60, gpl50, gpl40, gpl20 or mutants thereof. The DNA or MVA vectors could encode autologous and/or heterologous Env.

[113] In one embodiment, the method comprises c) administering DNA vector encoding EB354 T/F HIV Env, d) administering an MVA vector encoding EB354 T/F HIV Env, b) administering a boost with one or more of i) a recombinant gpl20 T/F HIV Env, ii) gpl20deltaN T/F HIV Env or iii) gpl40 SOSIP trimer HIV Env.

[114] In one embodiment, the method comprises

[115] administering a DNA vector comprising EB354 T/F Env,

[116] administering an MVA vector comprising EB354 T/F Env,

[117] administering a boost with recombinant MVA comprising EB354 2010 Env, 2014 Env, or 2015 Env,

[118] In a fifth aspect, a method is provided to broaden a broadly neutralizing antibody

response to HIV from an unmutated common ancestor comprising:

[119] a) administering a composition to prime the immune response comprising a vector

expressing VLPs displaying the native form of one or more Env proteins (Envs), wherein the Env proteins are capable of interacting with unmutated common ancestors (UCAs) for broadly neutralizing antibody (nAb) to initiate the Ab response, and

[120] b) administering a composition to boost the the primed immune response of a),

comprising a recombinant vector expressing VLPs displaying the same or different Env form as the priming Env administered in a), and

[121] c) administering one or more immunogenic vectors expressing mutated forms of the HIV Env protein that co-evolved with the generation of bnAb to broaden the primed immune response of a).

[122] In one embodiment the administration of immunogenic vectors broadens an initial nAb comprising mutated forms of a T/F Env that co-evolved with the generation of bnAb.

[123] In various embodiments the boosting vectors express gpl60 or various truncated forms of Env such as gpl50, gpl40, or gpl20.

[124] In one embodiment the boosting vectors are DNA vectors. [125] In one embodiment the boosting vectors are MVA vectors. [126] In one embodiment the HIV Envs are displayed on VLPs.

[127] In various embodiments boosting vectors express VLPs displaying Env sequences such as those from patient CH0505 T/F, week 53.16 Env, week 78.33 Env, and/or week 100.B6.

[128] In various embodiments boosting vectors express VLPs displaying Env sequences such as those from patient EB354 T/F, EB354 2010 Env, EB354 2014 Env, and/or EB354 2015 Env.

[129] In a sixth aspect, the invention provides a kit comprising a DNA prime-MVA boost immunogenic composition in which the DNA vector comprises a sequence encoding an HIV Env that binds with an UCA and the MVA vector boost expresses the same Env.

[130] In one embodiment, the priming DNA composition comprises a HIV Env protein displayed on VLPs that interacts with UCA for bnAb on B cell precursors and the boosting MVA composition comprises the same Env displayed on VLPs.

[131] In one embodiment, the priming DNA composition comprises the CH0505

transmitted/founder (T/F) Env protein that interacts with the V_H4-59 UCA for bnAb displayed on VLPs and the boosting MVA composition comprises the same Env displayed on VLPs. [132] In one embodiment, the boosting composition further comprises gpl20, pl20deltaN or gpl40 SOSIP trimers to enhance responses elicited by the native Envs displayed on VLPs by the DNA prime and MVA boost immunogenic compositions.

[133] In one embodiment the kit comprises an immunogenic vector construct for inducing a neutralizing antibody response to HIV-1 comprising (i) a vector expressing VLPs displaying Env capable of interacting with an UCA for bnAb and (ii) a composition to boost the primed response, comprising a second vector expressing VLPs displaying the same Env.

BRIEF DESCRIPTION OF THE DRAWINGS

[134] FIG. 1 A-C show DNA and MVA constructs and their expression. FIG. 1 A: Schematic for the expression cassette of the DNA vector expressing 505 T/F sequences. CMVIE+IA, CMV- immediate early promoter plus intron A. BGHpA, bovine growth hormone polyadenylation sequence. FIG. IB Schematic of MVA expressing MVA-T/F sequences. I8R and GIL, conserved vaccinia sequences flanking the insertion site for env; A50R and B1R, conserved sequences flanking the insertion site for gag. PmH5, a modified immediate early H5 vaccinia promoter. Numbers indicate positions in the MVA genome, which is abbreviated and not to scale. FIG. 1C Western blot of MVA-expressed Gag and Env proteins. Supernatants and cell lysates were collected from 293T cells infected with the indicated MVA viruses or mock infected (P), separated by SDS-PAGE, and western blotted for detection of Gag (p55) and uncleaved (gpl50) and cleaved (gpl20) Env. In (A) and (B) gpl20, HIV sequences encoding the receptor binding subunit of Env; gp41, HIV sequences encoding the transmembrane subunit of Env; gp41t, gp41 truncated at amino acid 36 of the endodomain; gag, HIV sequences encoding the group-specific antigens of HIV; x, inactivating point mutations in the zinc fingers for packaging of HIV RNA.

[135] FIG. 2 shows electron micrographs of VLPs expressed by the DNA and MVA vectors. Thin section electron micrographs were immunogold stained for Env using the PGT145 and PGT 151 recombinant Abs that bind native trimers. The DNA vector is expressed in transiently transfected 293T cells and the MVA vector in infected DFl cells. The VLPs being analyzed and nanometer (nm) size markers are indicated in the panels. Arrows, indicate examples of immunogold staining on VLPs. The triangles point to examples of immunogold staining on the plasma membranes of vector infected cells. [136] FIG. 3A-C shows antibody responses elicited by immunizations with the

transmitted/founder (T/F) immunogens. Four young adult rhesus were immunized at 0 and 8 weeks with 3 mg of DNA-T/F, boosted at weeks 16, 24, and 40 with lxlO⁸ Tissue Culture Infectious Doses to infect 50% of cultures (TCID50) of MV A- T/F and then boosted at week 56 with lxlO⁸ TCID50 of MVA-T/F plus 300 μg gpl20-T/F in 600 μg alhydrogel. FIG. 3A shows the median and ranges for binding Ab detected in an enzyme-linked immunosorbent assay (ELISA) for gpl20-T/F for the 4 macaques. FIG. 3B shows temporal patterns of binding Ab and neutralizing Ab for each of the macaques. Binding Ab is shown with a dashed line. Neutralizing Ab was determined using pseudoviruses with the tested Envs. The pseudovirus designated wk 4.3 has an easy to neutralize Tier 1 Env that appeared at 4.3 weeks of infection in patient CH0505. Neutralizing Ab for 505 T/F is for the Tier 2 transmitted/founder virus for patient CH0505. Neutralizing Ab for the CH0505TF.gly4 (gly4) and CH0505TF.gly3.276 (gly3) viruses are neutralizing activity for easy to neutralize mutants of the CH0505 T/F virus that detect precursors to neutralizing Ab for the CH0505 T/F virus. Neutralizing activity is measured as the dose that causes a 50% reduction in infection (ID50), FIG. 3C shows data mapping neutralizing activity using pseudoviruses with Env mutants that knock out the VI V2 target for bnAb (N160A and N160A.N173A); the CH0505 target for bnAb to the CD4bs (N280D and G458Y); and a V3glycan target for bnAb (N301A and N334A).

[137] FIG. 4A shows a map of shuttle plasmid pLW73-505env (gpl50 or gpl40). FIG. 4B shows annotated sequence (SEQ ID NO: 1) of plasmid pLW73-505Env wherein the Env is CH0505 T/F gpl50. Italic: Last 537 nt of MVA I8R gene; Italic and underlined: Last 229 nt of MVA I8R gene; underlined. Green Fluorescent Protein gene; Bold. mH5 vaccinia virus Promoter; Bold and underlined. CH505 transmitted founder Env (gpl50); Bold and Italic: Last 702 nt of MVA GIL gene. FIG. 4C shows the nucleotide sequence (SEQ ID NO: 2) of

Transmitted/Founder (T/F) CH505 Env sequence (gpl50). FIG. 4D shows the protein sequence (SEQ ID NO: 3) of Transmitted/Founder (T/F) CH505 Env sequence (gpl50). FIG. 4E shows the nucleotide sequence (SEQ ID NO: 4) of Week 53.16 CH505 Env (gp 150). FIG. 4F shows the protein sequence (SEQ ID NO: 5) of week 53.16 CH505 Env (gpl50). FIG. 4G, shows the nucleotide sequence (SEQ ID NO: 6) of week 78.33 CH505 Env sequence (gpl50). FIG. 4H, shows the protein sequence (SEQ ID NO: 7) of w78.33 CH505 Env sequence (gpl50). FIG. 41, shows the nucleotide sequence (SEQ ID NO: 8) of week 100.B6 505 CH505 Env (gpl50). FIG. 4J shows the protein sequence (SEQ ID NO: 9) of week 100.B6 505 CH505 Env (gpl50). FIG. 4K shows the nucleotide sequence (SEQ ID NO: 10) of Transmitted/Founder (T/F) CH505 Env sequence (gpl40).

[138] FIG. 4L shows the protein sequence (SEQ ID NO: 11) of Transmitted/Founder (T/F) CH505 Env sequence (gpl40)

[139] FIG. 4K and 4L show nucleic and amino acid sequence of Env CH0505 T/F gpl40. Based on the gpl40 design for the T/F Env, week 53.16, week 78.33, and week 100. B6 Envs could be designed as gpl40. These sequences including week 53.16 Env, the week 78.33 Env, and the week 100. B6 could be inserted in pLW73-505Env vector in place of T/F Env shown in FIG. 4 A and 4B. The nucleic acids are codon optimized for MVA expression. The sequence (between positions 1628 and 3838) from plasmid pLW73 that is inserted in the MVA vector is inserted 5' to 3' in a Smal site 3' to DR (MVA) in 18 gene and a Sail site 5' to Flank 2 GIL of MVA. The proprietary upstream "ATG" system is described in Vaccine 26 (2008): 486-493.

[140] FIG. 5A show a map of a shuttle plasmid pLW76-505Gag, which comprises an insert encoding CH0505T/F Gag protein. FIG. 5B shows annotated sequence of plasmid pLW76- 505Gag (SEQ ID NO: 12). The sequence (between positions 1578 and 3077) from plasmid pLW76 that is inserted in the MVA is inserted 5' to 3' in a Smal site 3' to DR(MVA) in DNA ligase and a Sail site 5' to Flank 2 in B l kinase of MVA. Italic: Last 504 nt on MVA DNA ligase gene; Italic and underlined: Last 192 nt on MVA DNA ligase gene; Underlined. Green Fluorescent Protein gene; Bold. mH5 vaccinia virus Promoter; Bold and underlined. Gag transmitted founder; Bold and Italic: Last 611 nt on MVA Bl kinase gene. FIG. 5C (SEQ ID NO: 13) and FIG. 5D (SEQ ID NO: 14) show the nucleic and amino acid sequences of the T/F Gag protein. The nucleic acid is codon optimized for MVA expression.

[141] FIG. 6A shows a map of DNA vector (DNA-T/F-gpl50) comprising CH505 T/F Env gpl50 sequence and CH505 T/F Gag sequence. FIG. 6B shows the sequence (SEQ ID NO: 15) and annotations for DNA-T/F-gpl50 of FIG. 6A. Using the map and sequence of the DNA vector, additional Env sequences including variant sequences from CH505 could be inserted in place of the gpl50 Env.

[142] FIG. 7A shows a map of DNA vector (DNA-T/F-gpl60) comprising CH505 T/F Env gpl60 sequence and CH505 T/F Gag sequence. FIG. 7B shows the sequence (SEQ ID NO: 16) and annotation for the DNA-T/F-gpl60 vector and insert of FIG. 7 A. Using the map and sequence of the DNA vector, additional Env sequences including variant sequences from

CH0505 could be inserted in place of the gpl60 Env.

DETAILED DESCRIPTION

[143] I. Definitions

[144] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise, e.g., "a peptide" includes a plurality of peptides. Thus, for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

[145] The term "antigen" refers to a substance or molecule, such as a protein, or fragment thereof, that is capable of inducing an immune response.

[146] The term "autologous neutralizing antibodies (anAb)" refers to Ab capable of neutralizing virus and close relatives of the virus with the Env used to generate Ab but not other viruses.

[147] The term "broadly neutralizing antibody (bnAb) B cell lineages" refers to B cell lineages for bnAb responses to HIV are determined by mapping the co-evolution of antibodies and founder virus to elucidate the evolutionary pathway of an antigen that lead to a bnAb response.

[148] Broadly neutralizing Ab (bnAb) refers to Ab capable of blocking infection by HIV isolates from different infected people and different clades.

[149] The term ""cell-mediated immune response" refers to the immunological defense provided by lymphocytes, such as the defense provided by sensitized T cell lymphocytes when they directly lyse cells expressing foreign antigens and secrete cytokines (e.g., IFN-gamma.), which can modulate macrophage and natural killer (NK) cell effector functions and augment T cell expansion and differentiation. The cellular immune response is the 2^nd branch of the adaptive immune response.

[150] The term "CH0505" refers to the HIV-infected individual who underwent infection with a transmitted/founder virus that generated bnAb to the CD4bs.

[151] The term "conservative amino acid substitution" refers to substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in substantially altered immunogenicity. For example, these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.

[152] The term "deletion" in the context of a polypeptide or protein refers to removal of codons for one or more amino acid residues from the polypeptide or protein sequence. The term deletion in the context of a nucleic acid refers to removal of one or more bases from a nucleic acid sequence.

[153] The term "directed lineage (D/L) immunizations for bnAb" refers to immunizations that use Env antigens along the evolutionary pathway to a bnAb response to drive the evolution of a bnAb response.

[154] The term "EB354" refers to the HIV-infected individual who underwent infection with a transmitted/founder virus that generated bnAb to the CD4bs.

[155] The term "Env" refers to an envelope protein encoded by a viral env gene.

[156] The term "fragment" in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment, a fragment of a full-length protein retains activity of the full-length protein. In another

embodiment, the fragment of the full-length protein does not retain the activity of the full-length protein. [157] The term "fragment" in the context of a nucleic acid refers to a nucleic acid comprising an nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 35 contiguous nucleotides, at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, at least 60 contiguous nucleotides, at least 70 contiguous nucleotides, at least contiguous 80 nucleotides, at least 90 contiguous nucleotides, at least 100 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 contiguous nucleotides, at least 175 contiguous nucleotides, at least 200 contiguous nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains activity of the full-length protein. In another embodiment, the fragment encodes a peptide or polypeptide that of the full- length protein does not retain the activity of the full-length protein.

[158] As used herein, the phrase "heterologous sequence" refers to any nucleic acid, protein, polypeptide or peptide sequence which is not normally associated in nature with another nucleic acid or protein, polypeptide or peptide sequence of interest.

[159] As used herein, the phrase "heterologous gene insert" refers to any nucleic acid sequence that has been, or is to be inserted into the recombinant vectors described herein. The

heterologous gene insert may refer to only the gene product encoding sequence or may refer to a sequence comprising a promoter, a gene product encoding sequence (such as GP, VP or Z), and any regulatory sequences associated or operably linked therewith.

[160] The term "homopolymer stretch" refers to a sequence comprising at least four of the same nucleotides uninterrupted by any other nucleotide, e.g., GGGG or TTTTTTT.

[161] The term "humoral immune response" refers to the stimulation of Ab production and the accessory proteins and events that accompany antibody production, including T helper cell activation and cytokine production, affinity maturation, and memory cell generation. The humoral immune response is one of two branches of the adaptive immune response.

[162] The term "humoral immunity" refers to the immunological defense provided by antibody, such as neutralizing Ab that can directly block infection; or, binding Ab that identifies a virus or infected cell for killing by such innate immune responses as complement (C')-mediated lysis, phagocytosis, and natural killer cells.

[163] The term "ID50" refers to the inhibitory dose of a nAb that causes a 50% reduction in infection.

[164] The term "immune response" refers to any response to an antigen or antigenic determinant by the immune system of a subject (e.g., a human). Exemplary immune responses include humoral immune responses (e.g., production of antigen-specific antibodies) and cell- mediated immune responses (e.g., production of antigen-specific T cells).

[165] The term "improved therapeutic outcome" relative to a subject diagnosed as infected with a particular virus (e.g., HIV) refers to a slowing or diminution in the growth of virus, or viral load, or detectable symptoms associated with infection by that particular virus; or a reduction in the ability of the infected subject to transmit the infection to another, uninfected subject.

[166] The term "inducing an immune response" means eliciting a humoral response (e.g., the production of antibodies) or a cellular response (e.g., the activation of T cells) directed against a virus (e.g., HIV) in a subject to which the composition (e.g., an immunogenic vector) has been administered.

[167] The term "insertion" in the context of a polypeptide or protein refers to the addition of one or more non-native amino acid residues in the polypeptide or protein sequence. Typically, no more than about from 1 to 6 residues (e.g. 1 to 4 residues) are inserted at any one site within the polypeptide or protein molecule.

[168] The term "modified vaccinia Ankara," "modified vaccinia ankara," "Modified Vaccinia Ankara," or "MVA" refers to a highly attenuated strain of vaccinia virus that can be used as a vector for viral nucleic acid sequence inserts. MVA is replication defective in mammalian cells but replication competent in chicken cells (where it underwent attenuation). MVA is reviewed in (Mayr, A. et al. 1975 Infection 3 :6-14; Swiss Patent No. 568,392).

[169] The term "neutralizing antibody" or "nAb" is meant an antibody which either is purified from, or is present in, a body fluid (e.g., serum or a mucosal secretion) and which recognizes a specific antigen and inhibits the effect(s) of the antigen in the subject (e.g., a human). As used herein, the antibody can be a single antibody or a plurality of antibodies.

[170] The term "neutralizing Ab (nAb)" refers to Ab capable of blocking a virus antigen that mediates entry into cells. [171] The term "non-neutralizing antibody" or "nnAb" refers to a binding antibody that is not a nAb.

[172] The term "prevent", "preventing" and "prevention" refers to the inhibition of the development or onset of a condition (e.g., an HIV infection or a condition associated therewith), or the prevention of the recurrence, onset, or development of one or more symptoms of a condition in a subject resulting from the administration of a therapy or the administration of a combination of therapies.

[173] The term "prophylactically effective amount" refers to the amount of a composition (e.g., the recombinant MVA vector or pharmaceutical composition) which is sufficient to result in the prevention of the development, recurrence, or onset of a condition or a symptom thereof (e.g., an HIV infection or a condition or symptom associated therewith or to enhance or improve the prophylactic effect(s) of another therapy.

[174] The term "recombinant" means a polynucleotide of semisynthetic, or synthetic origin that either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

[175] The term "recombinant," with respect to a viral vector, means a vector (e.g., a viral genome that has been manipulated in vitro, e.g., using recombinant nucleic acid techniques to express heterologous viral nucleic acid sequences.

[176] The term "regulatory sequence" "regulatory sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence. Not all of these control sequences need always be present so long as the selected gene is capable of being transcribed and translated.

[177] The term "shuttle vector" refers to a genetic vector (e.g., a DNA plasmid) that is useful for transferring genetic material from one host system into another. A shuttle vector can replicate alone (without the presence of any other vector) in at least one host (e.g., E. coli). In the context of MVA vector construction, shuttle vectors are usually DNA plasmids that can be manipulated in E. coli and then introduced into cultured cells infected with MVA vectors, resulting in the generation of new recombinant MVA vectors. [178] The term "silent mutation" means a change in a nucleotide sequence that does not cause a change in the primary structure of the protein encoded by the nucleotide sequence, e.g., a change from AAA (encoding lysine) to AAG (also encoding lysine).

[179] The term "subject" is means any mammal, including but not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, rats, mice, guinea pigs and the like.

[180] The term "surrogate endpoint" means a clinical measurement other than a measurement of clinical benefit that is used as a substitute for a measurement of clinical benefit.

[181] The term "surrogate marker" means a laboratory measurement or physical sign that is used in a clinical or animal trial as a substitute for a clinically meaningful endpoint that is a direct measure of how a subject feels, functions, or survives and is expected to predict the effect of the therapy (Katz, R., NeuroRx 1 : 189-195 (2004); New drug, antibiotic, and biological drug product regulations; accelerated approval— FDA. Final rule. Fed Regist 57: 58942-58960, 1992.)

[182] The term "surrogate marker for protection" means a surrogate marker that is used in a clinical or animal trial as a substitute for the clinically meaningful endpoint of prevention of HIV infection.

[183] The term "synonymous codon" refers to the use of a codon with a different nucleic acid sequence to encode the same amino acid, e.g., AAA and AAG (both of which encode lysine). Codon optimization changes the codons for a protein to the synonymous codons that are most frequently used by a vector or a host cell.

[184] The term "TCID50" refers to the tissue culture infectious doses of a virus required to infect 50% of cultures.

[185] The term "therapeutically effective amount" means the amount of the composition (e.g., the recombinant MVA vector or pharmaceutical composition) that, when administered to a mammal for treating an infection, is sufficient to effect such treatment for the infection.

[186] The term "Tier 1 isolates of HIV" refers to easy to neutralize, frequently laboratory adapted isolates of HIV.

[187] The term "Tier 2 isolates of HIV" refers to difficult to neutralize isolates of HIV characteristic of those undergoing transmission. [188] The term "TZM-bl" cells refers to an indicator cell line for HIV infection used in neutralization assays .

[189] The term "treating" or "treat" refer to the eradication or control of a HIV, a reduction in the titer of the HIV, a reduction in the numbers of HIV isolates that are transmitted, the reduction or amelioration of the progression, severity, and/or duration of a condition or one or more symptoms caused by the HIV resulting from the administration of one or more therapies, or the reduction or elimination of the subject's ability to transmit the infection to another, uninfected subject.

[190] The term "unmutated common ancestor (UCA)" refers to a germline Ig gene with potential to recognize the founder Env for initiation of a lineage to a specific Ab such as bnAb.

[191] The term "transmitted/founder (T/F) virus" refers to a virus that underwent transmission out of a swarm of HIV isolates that were present in a person transmitting the infection.

[192] The term "vaccine" means material used to provoke an immune response and confer immunity after administration of the material to a subject. Such immunity may include a cellular or humoral immune response that occurs when the subject is exposed to the immunogen after vaccine administration.

[193] The term "vaccine insert" refers to a nucleic acid sequence encoding a heterologous sequence that is operably linked to a promoter for expression when inserted into a recombinant vector. The heterologous sequence may encode a glycoprotein or matrix protein described here.

[194] The term "viral infection" means an infection by a viral pathogen (e.g., HIV) wherein there is clinical evidence of the infection based on symptoms or based on the demonstration of the presence of the viral pathogen in a biological sample from the subject.

[195] The term "virus-like particles" or "VLP" refers to a structure which resembles the native virus antigenically and morphologically.

[196] II. Vectors and Immunogenic Proteins

[197] Described herein are immunogenic expression vector and protein compositions and methods of use to elicit an immune response. In some embodiments, the response is a protective or therapeutic immune response to HIV. In some embodiments the response comprises inducing neutralizing antibodies, which could include broadly neutralizing antibodies. [198] The development of a safe, highly efficacious prophylactic HIV-1 vaccine is of paramount importance for the control and prevention of HIV-1 infection. A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) {Immunol. Rev. 254(2013): 225-244). (bnAbs) are neutralizing antibodies which neutralize multiple HIV-1 viral strains. bnAbs are unique in that they target conserved epitopes of the virus, meaning the virus may mutate, but the targeted epitopes will still exist. In contrast, non-bnAbs are specific for individual viral strains with unique epitopes.

[199] Passive transfer of bnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not induced by current vaccines. For the past 25 years, the HIV vaccine development field has used single or prime boost heterologous Envs as immunogens, but to date has not found a regimen to induce high levels of bnAbs. Immunogenic expression vectors and proteins have been employed to induce immune responses to pathogens.

[200] A new paradigm for design of strategies for induction of bnAbs employs B cell lineage immunogen design {Nature Biotech. 30(2012):423-33) in which the induction of bnAb lineages is recreated. B cell lineages are determined by mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction {Nature 496(2013): 469-76). From this type of work has come the hypothesis that bnAb induction will require (i) an antigen capable of stimulating the UCA for a bnAb and (ii) a selection of antigens to stimulate sequentially evolved nAbs that occur in the setting of bnAb generation in HIV infection {Nature 496(2013): 469-76).

[201] In certain aspects the invention provides compositions and immunization methods to elicit immune responses to HIV virus. In some embodiments the immune response comprises nAbs. In certain embodiments, the invention provides (i) immunogenic compositions used to initiate a response to HIV Envs that bind to UCA for bnAb and (ii) immunogenic compositions to broaden the response of the UCA to a bnAb response.

[202] A. MVA vector compositions

[203] Pox-based viral vectors including MVA vectors have been proposed for use in HIV immunization. See e.g. US Patent Publication 20150238593; U.S. Patent 7,795,017; US; U.S. Patent 8,623,379; Patent Publication 20150004132; U.S. Patents 7,867,982; U.S. Patent

9,453,239, and the following non-patent publications: Goepfert et al. Journal of Infectious Diseases 203 (2011): 610-619; Goepfert et al. Journal of Infectious Diseases, 210 (2014): 99- 110; Chamcha et al. Open Forum Infect Dis. 2016 Feb 1 l;3(l):ofw034. doi:

10.1093/ofid/ofw034. eCollection 2016; Iyer, Smita S., and Rama R. Amara. Vaccines 2.1 (2014): 160-178.

[204] To date, no study using nucleic acid or pox-based vectors expressing HIV Env has reported the induction of Tier 2 HIV Env nAb. The invention provides new and improved DNA and MVA based vectors comprising HIV Envs and methods for their use for induction of nAbs. In some embodiments the antibody responses comprise Tier 2 nAb to the HIV Env.

[205] In one embodiment, the recombinant DNA and MVA vectors comprise CH0505 HIV-1 Gag and Env immunogens, wherein the Env immunogens are displayed on VLPs or the plasma membranes of cells expressing the DNA and/or MVA vectors. In one embodiment, HIV Envs are selected based on binding to stages of the CHI 03 bnAb lineage: the CH505 transmitted founder (TF) and three natural CH505 variants (week 53, 78, and 100) (Bonsignori, M. et al., Cell 165, 449-463, April 7, 2016). The VLPs and production of the VLPs are intended to mimic a natural infection, stimulating both the humoral and cellular arms of the immune system to recognize, prevent and control the target infection should it appear.

[206] In various embodiments, any HIV gag sequence may be employed. Various HIV gag sequences, from various Clades, T/F viruses and mutants thereof are known in the art. In some embodiments, the DNA and MVA vectors of the invention do not comprise HIV polymerase as an immunogen. In some embodiments the HIV Env protein is one from an acute or chronic HIV infection or laboratory-generated mutant thereof. In some embodiments, the HIV Env protein or laboratory -generated mutant thereof elicits a protective immune response.

[207] In some embodiments, the Env is any one of CH0505 T/F Env, week 53.16 Env, week 78.33 Env, or week 100.B6 Env.

[208] In other embodiments, the Env is EB354 T/F Env, 2010 Env, 2014 Env or 2015 Env. (Freund et al., Sci Transl Med. 2017 Jan 18; 9(373): eaal2144)

[209] Two chronically HIV- infected individuals, RU1 and RU8, have developed VH1-46- derived bnAbs, 1B2530 and 8ANC131 (Scheid et al., 2011).

[210] In one embodiments, the Env is RU1 T/F HIV Env.

[211] In another embodiment, the Env is RU8 T/F HIV Env.

[212] In one embodiment, the viral vector comprises a sequence encoding an HIV Env protein displayed on VLPs or the plasma membrane of infected cells. In certain embodiments, the recombinant viral vector is a vaccinia viral vector, and more particularly, an MVA vector, comprising a sequence encoding the HIV Env protein. Vaccinia viruses have also been used to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines (Mackett, M. et al PNAS USA 79(1982):7415-7419; Smith, G. L. et al. Biotech Genet Engin Rev 2 (1984):383-407). This entails DNA sequences (genes) which code for foreign antigens being introduced, with the aid of DNA recombination techniques, into the genome of the vaccinia viruses. If the gene is integrated at a site in the MVA or DNA which does not disrupt an essential function for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious, that is to say able to infect foreign cells and thus to express the integrated DNA sequence (EP Patent Applications No. 83,286 and No. 110,385). The recombinant vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, on the other hand, for the preparation of heterologous proteins in eukaryotic cells.

[213] Several such strains of vaccinia virus have been developed to avoid undesired side effects of smallpox vaccination. Thus, a modified vaccinia Ankara (MVA) has been generated by long- term serial passages of the Ankara strain of vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A. et al. Infection 3(1975):6-14; Swiss Patent No. 568,392). The MVA virus is publicly available from American Type Culture Collection as ATCC No.: VR-1508. MVA is distinguished by its great attenuation, as demonstrated by diminished virulence and reduced ability to replicate in primate cells, while maintaining good immunogenicity. The MVA virus has been analyzed to determine alterations in the genome relative to the parental CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer, H. et al. J Gen Virol 72(1991): 1031-1038). The resulting MVA virus became severely host cell restricted to avian cells.

[214] Furthermore, MVA is characterized by its extreme attenuation. When tested in a variety of animal models, MVA was proven to be avirulent even in immunosuppressed animals. More importantly, the excellent properties of the MVA strain have been demonstrated in extensive clinical trials (Mayr A. et al. Zentralbl Bakteriol [B] 167(1978):375-390; Stickl et al. Dtsch Med Wschr 99(1974):2386-2392). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. [215] MVA replication in human cells was found to be blocked late in infection preventing the assembly to mature infectious virions. Nevertheless, MVA was able to express viral and recombinant genes at high levels even in non-permissive cells and was proposed to serve as an efficient and exceptionally safe gene expression vector (Sutter, G. and Moss, B. PNAS USA 89(1992): 10847-10851). Additionally, novel vaccinia vector vaccines were established based on MVA having foreign DNA sequences inserted at the sites such as for example but not limited to deletion III within the MVA genome (Sutter, G. et al. Vaccine 12(1994): 1032-1040).

[216] Recombinant MVA vaccinia viruses can be prepared as set out hereinafter. A DNA- construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a predetermined insertion site (e.g. between two conserved essential MVA genes such as I8R/G1L; in restructured and modified deletion III; or at other nonessential sites within the MVA genome) is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker. The DNA-construct to be inserted can be linear or circular. A plasmid or polymerase chain reaction product is preferred. Such methods of making recombinant MVA vectors are described in PCT publication WO/2006/026667 incorporated by reference herein. The DNA-construct contains sequences flanking the left and the right side of a naturally occurring deletion, or between two essential genes. The foreign DNA sequence is inserted between the sequences flanking the naturally occurring deletion or the two essential genes. For the expression of a DNA sequence or gene, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385). The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. Virol 52(1973):456-467; Wigler et al. Cell 16(1979):777- 785), by means of electroporation (Neumann et al. EMBO J. l(1982):841-845), by

microinjection (Graessmann et al. Meth Enzymol 101(1983):482-492), by means of liposomes (Straubinger et al. Meth Enzymol 101(1983):512-527), by means of spheroplasts (Schaffher PNAS USA 77(1980):2163-2167) or by other methods known to those skilled in the art. [217] In other embodiments MVA vectors comprising a sequence encoding the Env from an acute or chronic HIV infection or a mutant thereof that elicits a protective immune response. In one embodiment, the present invention is a recombinant viral vector (e.g., an MVA vector) comprising a sequence encoding the CH0505 transmitted/founder (T/F) Env protein.

[218] In some embodiments, the Env is any one of CH0505 T/F Env, week 53.16 Env, week 78.33 Env, or week 100.B6 Env.

[219] In one embodiment, the present invention is a recombinant viral vector (e.g., an MVA vector) comprising a sequence encoding the EB354 transmitted/founder (T/F) Env protein.

[220] In other embodiments, the Env is EB354 T/F Env, 2010 Env, 2014 Env or 2015 Env. (Freund et al., Sci Transl Med. 2017 Jan 18; 9(373): eaal2144)

[221] The viral vector (e.g., an MVA vector) may be constructed using conventional techniques known to one of skill in the art. The one or more heterologous gene inserts encode a polypeptide having desired immunogenicity, i.e., a polypeptide that can induce an immune reaction, cellular immunity and/or humoral immunity, in vivo by administration thereof. The gene region of the vector (e.g., an MVA vector) where the gene encoding a polypeptide having immunogenicity is introduced is flanked by regions that are indispensable to MVA growth. In the introduction of a gene encoding a polypeptide having immunogenicity, an appropriate promoter may be operatively linked upstream of the gene encoding a polypeptide having desired immunogenicity.

[222] In one embodiment, the deletion III site is restructured and modified to remove nonessential flanking sequences.

[223] In one embodiment, the MVA comprises 5 to 6 different sequences encoding HIV Env proteins.

[224] In one embodiment, the MVA comprises one HIV Gag and 4 Envs in the same MVA to produce VLPs expressing 4 different HIV Env proteins.

[225] In one embodiment, the MVA comprises a cocktail of vectors expressing different HIV Env proteins sharing a common lineage. The cocktail approach allows expression of a mix of VLPs with dedicated Envs. The cocktail is an appropriate approach for immunizing with several Envs. Dedicated Envs have been superior to particles with mixed Envs for immunization. The use of the large capacity could be to immunize for another protein or proteins that would not interfere with the VLP production. [226] In exemplary embodiments, the vector is constructed to express a sequence encoding the CH0505 transmitted/founder (T/F) Env protein), which is inserted between two conserved essential MVA genes (I8R and GIL) or into modified deletion site III using a shuttle vector.

[227] In exemplary embodiments, the vector is constructed to express a sequence encoding the EB354 transmitted/founder (T/F) Env protein), which is inserted between two conserved essential MVA genes (I8R and GIL) or into deletion site III using a shuttle vector.

[228] In certain embodiments, the polypeptide, or the nucleic acid sequence encoding the polypeptide, may have a mutation or deletion (e.g., an internal deletion, truncation of the amino- or carboxy-terminus, or a point mutation).

[229] The one or more genes introduced into the recombinant viral vector are under the control of regulatory sequences that direct its expression in a cell.

[230] In one embodiment, the sequence is inserted into deletion site I, II, III, IV, V or VI of the MVA vector.

[231] In one embodiment, the sequence is inserted between I8R and GIL of the MVA vector, or into restructured and modified deletion III of the MVA vector.

[232] In exemplary embodiments, the sequence may be, for example, under the control of a promoter selected from the group consisting of Pm2H5, Psyn II, or mH5 promoters.

[233] The present invention also extends to host cells comprising the recombinant viral vector described above, as well as isolated virions prepared from host cells infected with the

recombinant viral vector.

[234] In some embodiments, the vectors of the invention do not comprise an HIV polymerase as an immunogen. Exemplary non-limiting shuttle vectors used to construct the MVA vectors of the invention are described in US20120263750 Al and US Patent 9133478 B2 which contents are incorporated by reference in their entirety.

[235] In some embodiments, other Pox or MVA vectors could be used wherein the reagents and shuttle vectors described herein could be used to construct these additional vectors. Non-limiting examples of other constructions are described in Wyatt et al. Curr Protoc Mol Biol. (2017) 117 : 16.17.1 - 16.17.18. doi : 10.1002/cpmb .322004, incorporated by reference in its entirety .

[236] In some embodiments, there are alternative ways or positions in the MVA vector to insert the immunogens. In non-limiting examples the Gag and Env immunogens could be inserted in many other sites. These sites include spontaneous deletions that occurred during the attenuation of MVA and sites between essential genes. For non-limiting examples of the use of other sites, see Schweneker Journal of Virology, (2017) Mar 22. pii: JVI.00343-17. doi: 10.1128/JVI.00343- 17, incorporated by reference in its entirety

[237] B. DNA Vectors

[238] DNA vectors are provided that express membrane-bound HIV Env protein on VLPs and the plasma membranes of DNA-expressing cells. In other embodiments, the DNA vector expresses other HIV Env proteins from acute or chronic infections or laboratory-derived mutants of such that elicit protective immune responses. In one embodiment, the DNA vector expresses VLPs that display CH0505 HIV-1 Env proteins. In one embodiment, the DNA vector expresses VLPs that display EB354 HIV-1 Env proteins.

[239] Several exemplary plasmids have been previously constructed and used to express HIV antigens as VLPs (U.S. Patent 8,623,379). The expression plasmids constructed and used include pGAl and its derivatives pGAl . l and pGA1.2; and pGA2, and its derivatives pGA2.1 and pGA2.2. The immunogenic vector constructs are typically referred to with the "backbone" vector and the "insert" being separated by a dash or a backslash. These constructs express multiple HIV proteins by subgenomic splicing of a single RNA (Smith et al., Aids Research and Human Retroviruses 20 (2004): 654-665; Smith et al., Aids Research and Human Retroviruses 20 (2004): 1335-1347). Packaging of the RNA is minimized by the deletion of packaging sequences and by inactivating point mutations in the two zinc fingers in Gag as previously described (Smith et al., Aids Research and Human Retroviruses 20 (2004): 654-665; Smith et al.). Plasmids containing JS7-like inserts appear to exhibit better immunogenicity and are more efficient in priming an immune response (as evidenced by anti-Env antibodies) than plasmids containing JS2-like inserts. JS7 and JS2 differ in that JS7 has an inactivating point mutation in its protease gene (PR) (Aids Research and Human Retroviruses 20 (2004): 1335-1347). This mutation facilitates the formation of VLPs. While not to be bound by any theory, it is believed that the mutation precludes premature intracellular cleavage of the overexpressed pr55 Gag protein. Preclusion of premature cleavage of pr55 Gag can also be achieved by other mutations in PR or deletions of the HIV protease (PR). Accordingly, inserts that include inactivating mutations in PR, or no PR gene, such as in the pGAl-T/F HIV-1 DNA vector are a preferred embodiment for priming an immune response to HIV. In an exemplary embodiment, the vector is constructed to express a sequence encoding the CH0505 transmitted/founder (T/F) Env protein. [240] In certain embodiments, the polypeptide, or the nucleic acid sequence encoding the polypeptide, may have a mutation or deletion (e.g., an internal deletion, truncation of the amino- or carboxy-terminus, or a point mutation).

[241] The one or more genes introduced into the recombinant DNA vector are under the control of regulatory sequences that direct its expression in a cell. In exemplary embodiments, the sequence may be, for example, under the control of a promoter such as the cytomegalovirus immediate early promoter (CMVIE), the SV40 promoter, the desmin promoter or the creatine kinase promoter. Introns, such as intron A of the cytomegalovirus immediate early promoter (CMVIE) can be included to enhance expression of the plasmid vector insert. Transcriptional termination sequences can include such termination sequences as found for the bovine growth hormone sequence (BGH), the SV40 termination, or the rabbit beta-globin terminator.

Optimization of Kozak sequences can enhance expression as can codon optimization for human cells.

[242] In some embodiments, the DNA vectors of the invention do not comprise an HIV polymerase as an immunogen. The DNA vector may be constructed using conventional techniques known to one of skill in the art.

[243] C. Protein Boosts

[244] In one embodiment, a boosting composition is administered that comprises a gpl20 protein or gpl40 SOSIP trimer to enhance responses elicited by the native Envs displayed on VLPs by the DNA and MVA vectors. The gpl20 or gpl40 SOSIP trimer proteins can be delivered at the same time or different times than the DNA or MVA inoculations.

[245] In some embodiments of the invention the boosting composition can include a recombinant gpl20 protein, gpl20deltaN, or gpl40 SOSIP trimer, including without limitation a recombinant Env binding a germ -line Ig sequence for bnAb.

[246] There are other proteins known in the art that may be used as a protein boost in the methods described herein. Exemplary protein boosts are also described in US 20180036400, US 20140037681and US 20170290907.

[247] III: Pharmaceutical Compositions [248] The compositions can be formulated with appropriate carriers using known techniques to yield compositions suitable for various routes of administration. In certain embodiments the compositions are formulated with pharmaceutically acceptable carriers for intramuscular (IM), subcutaneous, intravenous, nasal, mucosal, or any other suitable route of administration.

[249] The pharmaceutical compositions may comprise one or more recombinant MVA vectors described herein, or one or more recombinant DNA vectors described herein and a

pharmaceutically acceptable carrier, wherein the one or more vectors comprises sequences encoding HIV immunogens are selected from HIV Gag, and HIV T/F Env.

[250] In certain embodiments, the methods and compositions comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies.

[251] In one embodiment, the pharmaceutical compositions comprise two or more recombinant MVA or DNA vectors encoding the same or different HIV Env proteins.

[252] In certain aspects the invention provides recombinant modified vaccinia Ankara (MVA) vectors comprising inserts encoding VLPs displaying HIV Env immunogens.

[253] In some embodiments the HIV Env is from other T/F viruses and lineages that were selected in infected cells for the elicitation of bnAb.

[254] In some embodiments the HIV Env protein is from an acute or chronic natural infection or a laboratory derived mutant of an HIV Env protein from an acute or chronic natural infection.

[255] In one embodiment, the HIV Env protein is capable of eliciting a protective immune response to HIV.

[256] In some embodiments the HIV Env is any one of the following CH0505 Envs: T/F, week 53.16, week 78.33, and week 100.B6.

[257] In some embodiments the HIV Env is any one of the following EB354 Envs: T/F, EB354 2010 HIV Env, EB354 2014 Env, and EB354 2015 Env.

[258] In some embodiments the vectors comprise additional sequences encoding immunogens, for example but not limited to HIV Gag, or any other suitable immunogen for priming or boosting an immune response to HIV.

[259] In some embodiments the pharmaceutical composition comprises a DNA prime-MVA boost immunogenic compositions wherein the DNA immunogenic composition comprises an HIV Env that binds with an UCA for bnAb and the boost comprises the same Env in an MVA vector.

[260] In some embodiments of the invention the UCA is V_H4-59 for bnAb to the CD4bs.

[261] In some embodiments of the invention the DNA or MVA expressed VLPs display Envs from infected individual CH0505.

[262] In some embodiments of the invention the expressed Env is the gpl60 form of the CH0505 transmitted/founder (T/F) Env.

[263] In some embodiments of the invention the expressed Env is a partially truncated Env such as a gpl50, gpl40, gpl20 Env or a mutant CH0505 T/F Env.

[264] In some embodiments of the invention the boosting composition can include a recombinant gpl20 protein, gpl20deltaN, or gpl40 SOSIP trimer, including without limitation a recombinant Env binding a germ -line Ig sequence for bnAb.

[265] In certain embodiments the composition comprises a selection of four CH0505 Envs for eliciting neutralizing Ab directed to the CD4bs— the T/F Env, the week 53.16 Env, the week 78.33 Env, and the week 100.B6 Env, wherein sequences encoding the Envs are inserted in DNA and/or MVA vectors, wherein in certain embodiments the Envs are produced in the vaccinated person and in some embodiments are displayed on VLPs.

[266] In one embodiment, the pharmaceutical composition comprises a DNA prime-MVA boost immunogenic compositions in which the DNA immunogenic composition comprises an HIV Env that binds with the UCA to a CD4bs lineage antibody and the boost comprises the same or different Env in an MVA vector. See Liao et al. Nature 496 (2013):469-476 including supplementary materials, and Bonsignori et al. Cell 165 (2016):449-63. doi:

10.1016/j .cell.2016.02.022. Epub 2016 Mar 3, and see also WO2014/042669 for CH0505 immunogens.

[267] In one embodiment, the priming DNA composition comprises a HIV Env protein displayed on VLPs that interacts with UCA for bnAb and the boosting composition comprises the same Env in an MVA, wherein the envelop is displayed on VLPs.

[268] In one embodiment, the priming DNA composition comprises the CH0505 transmitted/founder (T/F) Env protein that interacts with the V_H4-59 UCA for bnAb for the CD4bs and the boosting MVA composition comprises the same Env.

[269] In one embodiment, the boosting composition can include a gpl20 protein or gpl40 SOSIP trimer to enhance responses elicited by the native Envs displayed on VLPs by the DNA and MVA vectors. The gpl20, gpl20deltaN, or gpl40 SOSIP trimer proteins can be delivered at the same time or different times than the DNA or MVA inoculations.

[270] In some embodiments, the composition comprises a suitable pharmaceutically acceptable carrier.

[271] In one embodiment, the pharmaceutical composition comprises an adjuvant.

[272] The compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. The compositions can include an adjuvant, such as, for example but not limited to, alum, poly IC, MF-59 or other squalene-based adjuvant, ASOIB or other liposomal based adjuvant suitable for protein or nucleic acid immunization. In certain embodiments, TLR agonists are used as adjuvants. In other

embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions.

[273] In various embodiments, the adjuvant is selected from mineral gels such as aluminum hydroxide, aluminum salts (e.g., aluminum phosphate) or calcium salts (e.g., calcium phosphate); MF59, or SAF; adjuvant systems (AS01, AS02, AS03, AS04) (GlaxoSmithKline), complete Freund's adjuvant, incomplete Freund's adjuvant, microbially-derived adjuvants such as cholera toxin (CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72), Bacille Calmette-Guerin (BCG), lipopolysaccharides (LPS), mycobacteria, tetanus toxin, Corynebacterium parvum, DNA CpG motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate adjuvants such as immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres, or saponins (e.g., QS-21); cytokines such as IFN-γ, IL-1, IL-2, IL- 12 or GM-CSF; synthetic adjuvants such as nonionic block copolymers or surfactants, muramyl peptide analogues (e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine [thr-MDP], N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[l'- 2'-dipalmitoyl-s- n-glycero-3-hydroxyphosphoryloxy]-ethylamine), polyphosphazenes, synthetic polynucleotides, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, hydrocarbon emulsions, or keyhole limpet hemocyanins (KLH), CMC (carboxyl methylcellulose), HPMC (hydroxypropyl methylcellulose), glucopyranosyl Lipid adjuvant (GLA), or polylCLC.

[274] IV. Methods of Use

[275] The present invention relates to generation of antibody responses, including but not limited to Tier 2 nAb responses, to primary isolates of HIV. The present invention is based on the unexpected discovery that effective priming and boosting for neutralizing Ab to the CD4bs can be achieved using vector-expressed VLPs displaying the CH0505 T/F Env that binds the V_H4-59 UCA for bnAb to the CD4bs (McCurley et al. PLoS ONE 12(2017): e017786 or VLPs displaying the EB354 T/F Env. While not to be bound by theory, it is believed for CH0505 that trimeric CH0505 Envs on virus-like particles (VLPs) would present the same angle of approach to the CD4bs as the Envs in infected individual CH0505 that directed the generation of bnAb to the CD4bs from the germline immunoglobulin locus V_H4-59.

[276] In one embodiment, the methods described herein employ DNA-T/F and MVA-T/F CH0505 Env immunogens to elicit homologous Tier 2 neutralizing Ab to the CD4bs of the CH0505 T/F virus. Multiple immunizations with the VLP-displayed Envs were used to elicit homologous Tier 2 nAb to the CD4bs (see Fig. 3B). These multiple immunizations comprised priming with DNA and boosting with MVA, both of which displayed antigenically native CH0505 T/F Env on VLPs. A final boost included the recombinant gpl20 subunit of CH0505 Env to drive the breadth and height of primed Ab responses. In other embodiments, the recombinant protein boost could include gpl20, gpl20deltaN or gpl40 SOSIP trimer for the CH0505 T/F Env.

[277] In one embodiment, the methods described herein employ DNA-T/F and MVA-T/F EB354 Env immunogens to elicit homologous Tier 2 neutralizing Ab to the CD4bs of the EB354 T/F virus.

[278] Methods are provided for eliciting Ab responses to HIV targets for bnAb for the CD4bs. In this aspect a series of DNA or MVA-expressed Envs are used to initiate and then drive an immune response that has been initiated for an UCA for bnAb. The immunizing Envs are displayed as trimeric, Envs on the viral membranes of VLPs and plasma membranes of vaccine- expressing cells. The immunizing Envs can also be used as gpl20, gpl20deltaN or SOSIP trimer proteins. In this case boosting can be with heterologous as well as homologous Envs.

[279] In one embodiment, the generation of neutralizing Ab responses for primary isolates is accomplished by immunizing rodents or non-human primates with candidate immunogens, harvesting sera or plasma and testing for neutralizing Ab in assays conducted in cell cultures. One method for conducting these assays is the use of Env-defective viruses pseudotyped with the Envs of test viruses. Pseudovirions are analyzed for susceptibility to neutralization on the TZM- Bl indicator cell line. If neutralizing activity is scored, the neutralizing responses are mapped for specificity by testing pseudovirions with mutant Envs that abrogate specific targets for bnAb.

[280] In one embodiment, the method employs CH0505 Envs capable of binding to the V_H4-59 UCA for bnAb to the CD4bs, wherein the CH505 Envs are displayed as antigenically native Env on VLPs and the plasma membranes of vector-expressing cells. In some embodiments these VLPs give rise to epitopes recognized by the UCA.

[281] In one embodiment, the method employs EB354 Envs capable of binding to the V_H 4-4 and VL3-25, VH1-46/1-2 and VK3-20, or VH3-49 and Vkl-49 UCAs for bnAb to the CD4bs, wherein the CH505 Envs are displayed as antigenically native Env on VLPs and the plasma membranes of vector-expressing cells. In some embodiments these VLPs give rise to epitopes recognized by the UCA.

[282] In another embodiment, the one or more HIV immunogens are selected from HIV Gag, and any one of the following Envs: EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env.

[283] In another embodiment, the EB354 2010 Env, EB354 2014 Env, or EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[284] In some embodiments the UCA is V_H 4-4 and VL3-25, VH1-46/1-2 and VK3-20, or VH3-49 and Vkl-49 for bnAb to the CD4bs.

[285] In various embodiments, multiple immunizations with the VLP-displayed Envs are used to initiate a lineage and to drive the evolution of the Ab response to bnAb. These multiple immunizations consist of priming with DNA and boosting with MVA, both of which display antigenically native HIV Env. Boosts can also include the gpl20 subunit, gpl20deltaN, or gpl40 SOSIP trimers of an HIV Env to drive the breadth and height of primed antibody responses. The boosting Envs could be the same and/or different than the priming Env.

[286] In one embodiment, Envs capable of binding to an UCA for bnAb are used to raise bnAb to the epitopes recognized by the UCA. Multiple immunizations with the VLP-displayed Envs are used to initiate a lineage and drive the evolution of the Ab response to bnAb. These multiple immunizations consist of priming with DNA and boosting with MVA, both of which display antigenically native Env displayed on VLPs. Boosts can also include a gpl20 subunit, gpl20deltaN, or gpl40 SOSIP trimers of Env.

[287] In another aspect, the invention provides a method for eliciting a nAb response to a specific target on Env in a subject in need thereof comprising:

1) administering a first vector composition that expresses VLPs in the subject wherein the VLPs present an Env protein capable of binding to an UCA for initiating a bnAb immune response

2) administering a second vector composition presenting the same or different Envs on VLPs in the subject to drive the evolution and boost the immune response.

[288] VLPs can be expressed in the host being vaccinated by immunization with plasmid DNA or by infection with an infectious agent. In one embodiment, the first composition comprises a DNA vector expressing HIV antigens. In one embodiment, the first composition comprises an MVA vector expressing HIV antigens. In one embodiment, the second composition comprises an MVA vector expressing HIV antigens.

[289] In one embodiment, the Env encoded in the MVA or DNA vector and displayed on VLPs is the CH0505 T/F Env that binds to the V_H4-59 UCA for bnAb to the CD4bs. See Liao et al. Nature 496(2013):469-476. In one embodiment, protein boosts are used to enhance responses primed by the VLP-expressed Env.

In one embodiment, the Env encoded in the MVA or DNA vector and displayed on VLPs is the EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015 that binds to the V_H 4-4 and VL3-25, VH1-46/1-2 and VK3-20, or VH3-49 and Vkl-49 UCA for bnAb to the CD4bs. See Freund et al., Sci Transl Med. 2017 Jan 18; 9(373): eaal2144. In one embodiment, protein boosts are used to enhance responses primed by the VLP-expressed Env.

[290] In another embodiment, the EB354 2010 Env, EB354 2014 Env, and EB354 2015 Env bind bnAb NC37, BG1 and BG18 respectively.

[291] In one embodiment the initial nAb response to a target for bnAb is broadened by further boosting with Envs that co-evolved with the generation of bnAb. Non-limiting examples of such Envs from patient CH0505 are the week 53.16, week 78.33 and week 100. B6 Envs that were associated with nodes for the broadening of neutralizing activity. Non-limiting examples of such Envs from patient EB354 are the EB354 2010 Env, EB354 2014 Env, or EB354 2015 that were associated with nodes for the broadening of neutralizing activity. Boosting in some

embodiments comprises MVA vectors with these Envs. The boosting MVA vector could comprise the same Env as the priming vector. The boosting MVA vector could comprise a different Env. Boosting in some embodiments comprises addition of homologous or

heterologous recombinant protein, e.g. but not limited to gpl20 Env, gpl20deltaN, or gpl40 SOSIP trimers.

[292] V. Routes of Administration

[293] A DNA vector, an MVA vector or a boosting protein composition such as gpl20 protein as described herein may be administered by any appropriate route. In some embodiments, the composition is administered parenterally. In some embodiments, the parenteral administration is selected from intravenous, intradermal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, and/or transmucosal administration. In some embodiments, a composition as described herein is administered subcutaneously. For example, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, a composition as described herein is administered intravenously. More than one route can be used concurrently, if desired.

[294] The compositions utilized in the methods described herein can be administered by a route selected from, e.g., parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, periungual, nasal, rectal, topical administration, and oral administration. Parenteral

administration includes intravenous, intraperitoneal, subcutaneous, intraarterial, intravascular, and intramuscular administration. The method of administration can vary depending on various factors (e.g., the components of the composition being administered and the age of the person being immunized).

[295] Administration of the pharmaceutical compositions of the present invention can be by any of the routes known to one of skill in the art. Administration may be by, e.g., intramuscular injection. The compositions utilized in the methods described herein can also be administered by a route selected from, e.g., parenteral, dermal, transdermal, ocular, inhalation, buccal, sublingual, periungual, nasal, rectal, topical administration, and oral administration. Parenteral

administration includes intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The method of administration can vary depending on various factors, e.g., the components of the composition being administered and the severity of the condition being treated.

[296] In addition, single or multiple administrations of the compositions of the present invention may be given to a subject. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, e.g., measuring amounts of neutralizing secretory and serum antibodies. The dosages may then be adjusted or repeated as necessary to maintain desired levels of protection against viral infection.

[297] VI. Dosing Schedules

[298] Various embodiments may include differing dosing regimens. In various embodiments, the compositions described herein are administered at intervals of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, and greater than 12 weeks. A skilled artisan can readily determine the dosing and timing of immunizations.

[299] It will also be appreciated that single or multiple administrations of the immunogenic compositions of the present invention may be carried out. Levels of induced immunity can be monitored by measuring amounts of binding and neutralizing secretory and serum antibodies as well as levels of T cells, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection.

[300] In one embodiment, administration is repeated at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or more than 8 times. [301] In one embodiment, administration is repeated twice, three times, four times, five times, six time, seven times, eight times or more than eight times.

[302] In one embodiment, about 2-8, about 4-8, or about 6-8 administrations are provided.

[303] In one embodiment, about 1-4-weeks, 2-4 week, 3-4 week, 1 week, 2 week, 3 week, 4 week or more than 4 week intervals are provided between administrations.

[304] In one specific embodiment, a 4-week interval is used between 2 administrations.

[305] In one embodiment, a DNA vector is administered once and then again after 8 weeks (DD).

[306] In one embodiment, a MVA vector with or without protein compositions are

administered multiple times such as at weeks 16, 24 and 40 after initial DNA prime (MMM or M+P, M+P, M+P).

[307] In one embodiment, a single administration regimen is used for a combination composition comprising vectors expressing 4 or 5 HIV Env proteins of a bnAb lineage.

[308] In one embodiment, one administration regimen comprises administering vectors encoding CH0505 T/F HIV Env, week 53.16 HIV Env, week 78.33 HIV Env and week 100.B6 HIV Env.

[309] In one embodiment, one administration regimen comprises administering vectors encoding EB354 T/F HIV Env, 2010 HIV Env, 2014 HIV Env and 2015 HIV Env.

[310] In one embodiment, an administration regimen is used for each HIV Env protein. Such a regimen promotes the generation of long-lasting B-cells responsive to the immunogen for a robust immune response, specifically a robust antibody response.

[311] In one embodiment, a series of five administration regimens are performed for vectors encoding CH0505 T/F HIV Env, week 53.16 HIV Env, week 78.33 HIV Env and week 100.B6 HIV Env.

[312] In one embodiment, a series of four administration regimens are performed for vectors encoding EB354 T/F HIV Env, 2010 HIV Env, 2014 HIV Env and 2015 HIV Env.

[313] Dosing of proteins and nucleic acids can be readily determined by a skilled artisan. A single dose of nucleic acid can range from a few micrograms ^g) to several milligrams of a single immunogenic nucleic acid. Recombinant protein dose can range from a few μg micrograms to a few hundred micrograms, or milligrams of a single immunogenic polypeptide.

[314] When employed for the methods described herein, the compositions of the present invention are administered in the form of pharmaceutical compositions formulated for administration to a subject in pharmaceutically acceptable carriers. These pharmaceutically acceptable compositions represent further embodiments of the present invention.

[315] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be monitored on a patient-by-patient basis. However, suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 5.0 ^χ 10⁷ TCID₅₀ to about 5.0 x 10⁸ TCID₅₀. The dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight, and the nature of the formulation.

[316] For the priming compositions, one can arrive at an appropriate dosage when delivering DNA by way of a vector, just as one can when an MVA vector is used.

[317] In one embodiment, between about 100 μg to 5 mg of a DNA vector, is administered intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

[318] In other embodiments, between about 100 μg to 4 mg, between about 100 μg to 3 mg, between about 100 μg to 2 mg, between about 100 μg to 1 mg, between about 200 μg to 5 mg, between about 300 μg to 5 mg, between about 400 μg to 5 mg of a DNA vector, between about 500 μg to 5 mg of a DNA vector, between about 600 μg to 5 mg of a DNA vector, between about 700 μg to 5 mg of a DNA vector, between about 800 μg to 5 mg of a DNA vector, between about 900 μg to 5 mg of a DNA vector, between about 1 mg to 5 mg of a DNA vector, between about 2 mg to 5 mg of a DNA vector, between about 3 mg to 5 mg of a DNA vector, between about 4 mg to 5 mg of a DNA vector, between about 100 μg to 4 mg, between about 200 μg to 4 mg, between about 300 μg to 4 mg, between about 400 μg to 4 mg of a DNA vector, between about 500 μg to 4 mg of a DNA vector, between about 600 μg to 4 mg of a DNA vector, between about 700 μg to 4 mg of a DNA vector, between about 800 μg to 4 mg of a DNA vector, between about 900 μg to 4 mg of a DNA vector, between about 100 μg to 3 mg, between about 200 μg to 3 mg, between about 300 μg to 3 mg, between about 400 μg to 3 mg of a DNA vector, between about 500 μg to 3 mg of a DNA vector, between about 600 μg to 3 mg of a DNA vector, between about 700 μg to 3 mg of a DNA vector, between about 800 μg to 3 mg of a DNA vector, between about 900 μg to 3 mg of a DNA vector, between about 100 μg to 2 mg, between about 200 μg to 2 mg, between about 300 μg to 2 mg, between about 400 μg to 2 mg of a DNA vector, between about 500 μg to 2 mg of a DNA vector, between about 600 μg to 2 mg of a DNA vector, between about 700 μg to 2 mg of a DNA vector, between about 800 μg to 2 mg of a DNA vector, or between about 900 μg to 2 mg of a DNA vector is administered intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

[319] In one embodiment, about lxlO⁸ TCID50 of an MVA vector, is administered

intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

[320] In other embodiments, between about lxlO⁷ TCID50 and about lxlO⁹ TCID50, between about lxlO⁷ TCID50 and about lxlO⁸ TCID50 of an MVA vector, is administered intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

[321] In one embodiment, the MVA vector is administered at lxlO⁸ TCID50.

[322] In one embodiment, about 300 μg of HIV Env protein is administered as a boost. In one embodiment, the boosting composition can include a gpl20 protein, a gpl20deltaN or gpl40 SOSIP trimer to enhance responses elicited by the native Envs displayed on VLPs by the DNA and MVA vectors. The gpl20, gpl20deltaN or gpl40 SOSIP trimer proteins can be delivered at the same time or different times than the DNA or MVA inoculations.

[323] In one embodiment, between about 100 μg and about 600 μg of HIV Env protein, about 100 μg and about 500 μg of HIV Env protein, about 100 μg and about 400 μg of HIV Env protein, about 100 μg and about 300 μg of HIV Env protein, about 100 μg and about 200 μg of HIV Env protein, about 200 μg and about 600 μg of HIV Env protein, about 300 μg and about 600 μg of HIV Env protein, about 400 μg and about 600 μg of HIV Env protein, about 500 μg and about 600 μg of HIV Env protein, about 200 μg and about 500 μg of HIV Env protein, about 300 μg and about 400 μg of HIV Env protein, is administered intramuscularly, intradermally, intravenously, subcutaneously or mucosally.

[324] In one embodiment, the HIV Env protein dosage is between about 100 to 300 μg in an alum adjuvant. The alum adjuvant can be an aluminum phosphate or an aluminum hydroxide. Formulations can also be in other adjuvants, although alum is favored because protective immune responses were observed using this adjuvant in the partially effective RV144 trial. [325] In one regimen, DNA priming composition is administered at a dose of 250 μg up to 4 mg/injection, followed by MVA at a dose of 10⁷ to 10⁹ TCID50 per injection.

[326] The compounds of the invention are useful in inducing or boosting an immune response to HIV and dosage amounts can be determined by routine methods of clinical testing to find the optimum dose.

[327] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application is specifically and individually indicated to be incorporated by reference.

[328] The compositions and methods described herein may be further illustrated by the following non-limiting examples.

EXAMPLES

[329] Example 1: CH0505 Envs

[330] Sequences for HIV-1 Envs and antibodies to the HIV-1 Env from HIV-1 infected individual CH0505 were isolated as described in Liao et al. Nature 496(2013):469-476 including supplementary materials, and Bonsignori et al. Cell. (2016) Apr 7; 165(2):449-63. doi:

10.1016/j .cell.2016.02.022. Epub 2016 Mar 3, and see also WO2014/042669 for CH505 immunogens. The terms CH0505 and CH505 are used interchangeably.

[331] Any form of the Env is contemplated for use in the invention: gpl20, gpl20deltaN (see WO2014/042669), gpl40, gpl45, gpl50, gpl60.

[332] Recombinant HIV-1 proteins

[333] HIV-1 Env genes for subtype B, 63521, subtype C, 1086, and subtype CRF_01, 427299, as well as subtype C, CH0505 T/F Env were obtained from acutely infected HIV-1 subjects by single genome amplification, codon-optimized using the codon usage of highly transcribed human genes, de novo synthesized (GeneScript) as gpl60, gpl50, gpl40 or gpl20 (AE.427299) and cloned into a mammalian expression plasmid pcDNA3.1/hygromycin (Invitrogen).

Recombinant Env glycoproteins were produced in 293F cells cultured in serum-free medium and transfected with the HIV-1 gpl40- or gpl20-expressing pcDNA3.1 plasmids, purified from the supernatants of transfected 293F cells by using Galanthus nivalis lectin-agarose (Vector Labs) column chromatography, and stored at -80 °C. Selected Envs, such as the CH0505

transmitted/founder Env were further purified by superose 6 column chromatography to trimeric forms, and used in binding assays that showed similar results as with the lectin-purified oligomers.

[334] Example 2: Construction of DNA and MVA vectors expressing VLPs displaying

CH0505 Envs. VLP-expressing DNA and MVA vectors were constructed using the CH0505 Env sequences (McCurley et al. PLoS ONE (2017)12(10): e0177863.

https://doi.org/10.1371/journal.pone.0177863). Construction of the DNA vector used synthetic DNA sequences whereas the construction of the MVA vectors used synthetic shuttle vectors and a parental MVA that had been harvested in 1974 before the appearance of bovine spongiform encephalopathy (BSE) and sent in 2001 to Dr. Bernard Moss at NIAID, where it was plaque purified 3 times using certified reagents from sources free of BSE. Both vectors were constructed using standard techniques.

[335] The DNA vector used the pGAl expression vector (Smith et al. Aids Research and Human Retroviruses 20(2004):654-655) to express Gag, Tat, Rev, Vpu and Env by subgenomic splicing of a single RNA (Figure 1 A). Packaging of viral RNA was minimized by the deletion of packaging sequences found in the 5' untranslated region of HIV and by inactivating point mutations in the two zinc fingers in Gag as previously described (Smith et al. Aids Research and Human Retroviruses 20(2004):654-655). The DNA vector was designated DNA-T/F (McCurley et al. PLoS ONE (2017)12(10): e0177863. https://doi.org/10.1371/journal.pone.0177863).

The MVA vectors were constructed using the pLW76 shuttle vector to place T/F gag sequences in a modified and restructured insertion site III and the pLW73 shuttle vector to insert env sequences between two essential vaccinia genes (I8R and GIL) (Wyatt et al. J. Virol. 83 (2009):7176-84)(Figure IB). For the MVA vectors, Env sequences were truncated for nucleotides encoding the 115 C-terminal amino acids of the endodomain of gp41 to eliminate three previously identified endocytic sequences within gp41 that reduce display of Env on VLPs and the plasma membranes of infected cells (Wyatt et al. Virology 372(2007):260-272). Gag and Env inserts were optimized for the codon usage of vaccinia virus and sequences encoding termination of vaccinia transcripts were eliminated by using alternate codons (Wyatt et al. J. Virol. 83 (2009):7176-84). The shuttle vectors use the modified H5 early/late promoter to drive transcription (Wyatt et al. Vaccine 14 (1996): 1451-1458. The pLW73 Env expression cassette was modified to include a proprietary upstream ATG that reduces Env expression to levels that allow better processing of the overexpressed Env as demonstrated by more complete proteolytic cleavage of the gpl50 precursor into gpl20 and gp30 subunits. The rMVA vectors were designated MVA-T/F, MVA53C, MVA78C, and MVA100C (McCurley et al. PLoS ONE (2017)12(10): e0177863. https://doi.org/10.1371/journal.pone.0177863).

Western blots were used to assess the expression of Gag and Env (Figure 1C). Gag, which was expressed in the absence of protease, was present as uncleaved pr55 in both cell lysates and supernatants. Env was present as uncleaved gpl60 (for DNA vectors), or gpl50 (for MVA vectors) as well as cleaved gpl20 forms in cell lysates and almost exclusively as the mature gpl20 form in supernatants. Consistent with the formation of budding VLPs, temporal studies showed expressed protein moving from being primarily detected in cell lysates to being predominantly detected in cell supernatants.

[336] Example 3: Demonstration of VLP expression displaying native Env. Immuno- electron microscopy was used to test for the DNA and MVA vectors expressing VLPs displaying native Env (McCurley et al. PLoS ONE (2017)12(10): e0177863.

https://doi.org/10.1371/journal.pone.0177863) (Figure 2). Cells were plated on poly-D-lysine- treated Aclar discs (Electron Microscopy Sciences) in 12-well tissue culture plates. For expression of VLPs by DNA, 293T cells were transfected with 0.5 μg of DNA-T/F. For expression of VLPs by the MVA vectors, DF-1 cells, a continuous chicken cell line, were infected at a MOI of 1 with MVA-T/F, MVA53C, MVA78C, or MVA100C. Two-days later, vectors-expressing cells were incubated for 2 hours at 37° with a mixture of 10 μg/ml of trimer- specific recombinant Ab PGT 143 (specific to VI V2 tip) and PGT 151 (specific go the cleaved gpl20-gp41 interface) and then washed several times with culture medium. Cells were then fixed with 1% glutaraldehyde in 0.1 M phosphate buffer (pH7.4) and delivered to the Emory University Robert P. Apkarian Integrated Electron Microscopy Core for incubation with 6 nm colloidal gold particle conjugated goat anti-human secondary antibody. Upon finishing antibody incubation, cells were further fixed with 2.5% glutaraldehyde, and post fixed with 1% osmium tetroxide. Cells were then dehydrated with increasing percent solutions of ethanol and embedded in Eponate 12 resin. Ultrathin sections were cut at 70-80 nm thicknesses, and stained with 5% uranyl acetate and 2% lead citrate. Cell imaging was done on a JEOL JEM-1400 transmission electron microscope (JEOL Ltd) equipped with a Gatan US 1000 CCD camera (Gatan). The immunoelectron microscopy clearly demonstrated that the cells transfected with the DNA-T/F or infected with the MVA-T/F, MVA53C, MVA78C, or MVA100C vectors produced VLPs displaying native forms of CH0505 Envs (Figure 2) (McCurley et al. PLoS ONE (2017)12(10): e0177863. https://doi.org/10.1371/journal.pone.0177863). Env display was observed on released VLPs, budding VLPs and plasma membranes (Figure 2). Staining for Env was more frequent on VLPs produced by the MVA vectors than the DNA vector (Figure 2). This is consistent with the gpl50 form of Env expressed by the MVA vectors undergoing higher levels of surface expression and incorporation into virus than the gpl60 form expressed by the DNA (Wyatt et al. Virol.372 (2008):260-72).

[337] Example 4: First immunogenicity study. An immunogenicity study for the DNA-T/F and MVA-T/F vectors was conducted to determine if they could elicit homologous nAb for the CD4bs of the CHAVI 0505 T/F virus. Such nAb represented the first step in the lineage for elicitation of bnAb in patient CHAVI 0505 (Liao et al. Nature 496(2013):469-476 including supplementary materials). The 0505 DNA-T/F and MVA-T/F vectors were tested in two male and two female 4-6 years old rhesus macaques. The study was conducted at the New Iberia Research Center, Lafayette, Louisiana in accordance with all rules of the American Association for Laboratory Animal care and under the supervision of the Institutional Animal Clinical Care and Use Committee. Priming was with 3 mg of the DNA-T/F (D) vector inoculated by needle and syringe into the left thigh at weeks 0 and 8. Boosting was with lxlO⁸ TCID50 of MVA-T/F (M) vectors inoculated intramuscularly into the right thigh at weeks 16, 24 and 40 followed by a final boost at 56 weeks with lxlO⁸ TCID50 of MVA-T/F in the right thigh and 300 μg of gpl20- T/F formulated in 600 μg of alhydrogel (Brenntag Biosector, CAS 21645-51-2)(M+P) into the left thigh. Sera were collected at regular intervals throughout the trial. Lymph nodes as well as an exsanguination bleed were collected at the end of the trial, 2 weeks following the last boost. The general health, weights, clinical blood counts (CBC) and clinical chemistries of animals were normal throughout the trial.

[338] Example 5. Elicitation of binding Ab for gpl20-T/F . Binding Ab titers to CH0505 gpl20- T/F were assessed by Enzyme-linked-immunosorbent-assays (ELISA) (Figure 3 A). CH0505 gpl20-T/F was produced in 293F cells at the Duke Human Vaccine Institute. Assays included a standard curve of macaque IgG captured by goat anti-rhesus Ab and results were interpolated to estimate μ of specific Ab per ml. Binding Ab for the T/F gpl20 protein rose with immunizations one to four following which it increased and contracted with immunizations (Figure 3A). At peak levels, Ab reached hyperimmune titers estimated as high as 3.1 mg per ml and as low as 1.5 mg per ml. The troughs for binding Ab at the time of the final boost ranged from 0.1 to 0.4 mg per ml. Overall, the inclusion of protein in the final boost increased the titers of binding Ab from 5 to 31 -fold over the peak response elicited by the penultimate MVA boost

[339] Example 6. Elicitation Of Autologous Tier 2 Neutralizing Ab To The CD4bs For The CH0505 T/F Virus. Tests for neutralizing Ab to the CH0505 T/F virus (T/F) revealed the presence of autologous neutralizing Ab for the CD4bs in 2 of 4 rhesus (Figure 3B). In one macaque (Al 1R082) low titer neutralizing Ab (29 ID50) was transiently detected after the 5^th immunization. Evidence that this neutralization activity was real came from more durable (detected after the 5^th and 6^th immunizations) and higher titer activity (ID50 of 44-48) against the CH0505TF.gly3.276 and CH0505TF.gly4 (gly4) mutants. These glycan-deleted Tier 2 viruses are highly sensitive to CD4bs antibodies including the precursors to the bnAbs for the CD4bs of the CH0505 T/F virus (Figure3B). In the 2^nd animal, Al 1L068, higher titer neutralizing Ab for the 505 T/F virus (ID50 of 152 and 219 in independent assays) appeared post the 6^th

immunization, which included the 1^st gpl20 protein boost (Figure3B). This animal had relatively high titers of neutralizing Ab against the Tier IB CH0505TF.gly3.276 (gly3) and CH0505TF.gly4 (gly4) mutants by the 4^th immunization (ID50 of 334 to 2057). In Al 1L068 the CD4bs neutralizing activity achieved a plateau of 100% neutralization.

Neutralizing Ab for the easy to neutralize Tier 1 virus present in patient CH0505 at week 4.3 (4.3) post infection was dtected in all 4 test animals by the 2^nd MVA immunization (Figure 3B). These titers ranged from an ID50 of 2068 in A10L002 to an ID50 of 47,000 in Al 1R082.

[340] Assays for neutralizing Ab tested for neutralizing activity on TZM-bl cells using pseudovirions for the CH0505 lineage Envs (all Tier 2) plus the Env of the Tier 1 virus that appeared at week 4.3 post infection in the CH0505 patient (Sarzotti-Kelso et al. J. Immunol Methods 20\4 July ; 0: 131-146. doi: 10.1016/j .jim.2013.1 1.022).

[341] Example 7. Mapping of the neutralizing activity for the CH0505T/F virus to the CD4bs. The neutralizing Ab were mapped to the CD4bs using CH0505 pseudoviruses with inactivating point mutations in targets for bnAB. Inactivating mutations in the VI V2 glycan site (N160A and N160A.N173A) and the V3 glycan site (N301A and N334A) had no effect on neutralization (Figure4C). In contrast, mutations that knock out binding to the CH0505 CD4bs (N280D and G458Y) abrogated the neutralizing activity. Plasma neutralizing activity was not enhanced by mutations that increase neutralization by precursors to the CD4-mimicking bnAb VRCOl suggesting that neutralizing activity did not use the same CD4bs epitopes as VRC01.

[342] Example 8, Second immunogenicity trial and comparison of gpl20deltaN and gpl40 SOSIP trimers for boosting homologous Tier 2 nAB for the CD4bs of the CH0505 T/F virus. A 2^nd immunogenicity study is ongoing to extend the results in the 1st study and compare the effect of two different protein boosts on eliciting nAb for the CD4bs. This study is being conducted at Bioqual, Inc. in 6 young adult rhesus macaques. Higher and more frequent vector doses delivered to more than one site (for example each limb) are being tested for their ability to expedite the initiation of B cell lineages producing nAb for the CD4bs of the CH0505 T/F virus. In the 1^st study, immunizations were delivered over one year. In the 2^nd study they are being delivered over a period of 6 months. The 2^nd study also includes no DNA prime and a 10-times higher dose of MV A- T/F (lxlO⁹ TCID50) to initiate the lineage. The 2^nd study also compares the effects of gpl20deltaN and gpl40 SOSIP trimers for boosting nAb to the CD4bs of the CH0505 T/F virus.

[343] Table 1. Exemplary embodiment of the 2^nd immunization schema¹

Claims

What is claimed is:

1. A recombinant modified vaccinia Ankara (MVA) vector comprising a nucleic acid insert encoding one or more HIV immunogens that elicit an immune response against an HIV virus wherein the insert comprises an HIV gag sequence, and a sequence encoding HIV Env, or lineage-related HIV Env proteins of a HIV T/F Env.

2. The recombinant MVA vector of claim 1, wherein the vector comprises a nucleic acid insert encoding one or more HIV Env proteins, wherein the one or more HIV Env proteins are (i) identified from an acute or chronic natural infection or (ii) a laboratory-derived mutant of an HIV Env protein identified from an acute or chronic natural infection.

3. The recombinant MVAvector of claim 2, wherein the HIV Env protein or mutant HIV Env protein elicits a broad neutralizing antibody (bnAb) response to HIV.

4. The recombinant modified vaccinia Ankara (MVA) vector of claim 1 wherein the HIV Env is selected from the group consisting of CH0505 T/F Env, CH0505 week 53.16 Env, CH0505 week 78.33 Env, or CH0505 week 100.B6 Env.

5. The recombinant MVA vector of claim 1, wherein the HIV gag sequence comprises SEQ ID NO: 13 (CH0505 T/F gag) and the HIV Env is encoded by SEQ ID NO: 2 (CH0505 T/F gpl50 env).

6. The recombinant MVA vector of claim 1, wherein the HIV gag sequence comprises SEQ ID NO: 13 (CH0505 T/F gag) and the HIV Env is encoded by SEQ ID NO: 4 (CH0505 week 53.16 env).

7. The recombinant MVA vector of claim 1, wherein the HIV gag sequence comprises SEQ ID NO: 13 (CH0505 T/F gag) and the HIV Env is encoded by SEQ ID NO: 6 (CH0505 week 78.33 env).

8. The recombinant MVA vector of claim 1, wherein the HIV gag sequence comprises SEQ ID NO: 14 (CH0505 T/F gag) and the HIV Env is encoded by SEQ ID NO: 8 (CH0505 week 100.B6 env).

9. The recombinant MVA vector of claim 1, wherein the HIV gag comprises HIV EB354 gag and the HIV Env is selected from the group consisting of: EB354 T/F Env, EB354 2010 Env, EB354 2014 Env, or EB354 2015.

10. The recombinant MVA vector of claim 9, wherein the EB354 2010 Env, EB354 2014 Env and EB354 2015 Env bind to bnAb NC37, BG1 and BG18 respectively.

11. The recombinant MVA vector of claim 1, further comprising a promoter operably linked to the nucleic acid insert such that cellular expression of the HIV gag and HIV Env produces virus-like particles (VLPs) displaying Env.

12. A recombinant deoxyribonucleic acid (DNA) vector comprising an insert comprising consecutive nucleic acids comprising an HIV gag sequence, and a sequence encoding a HIV T/F Env, or a lineage-related HIV Env protein of the HIV T/F Env, wherein the one or more HIV immunogens elicit an immune response against an HIV virus.

13. The recombinant DNA vector of claim 12, wherein the HIV Env is (i) identified from an acute or chronic natural infection or (ii) a laboratory-derived mutant of an HIV Env protein identified from an acute or chronic natural infection.

14. A pharmaceutical composition comprising one or more of the vectors of claim 1 or claim 12 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition comprises two or more recombinant MVA or DNA vectors comprising inserts encoding the same or different HIV Env proteins.

16. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition further comprises (i) a protein boost selected from the group consisting of recombinant gpl20 protein, gpl20deltaN, a gpl40 SOSIP trimer, and/or (ii) a recombinant Env capable of binding a germ-line Ig sequence for bnAb.

17. The pharmaceutical composition of claim 14, wherein the composition further comprises a suitable adjuvant.

18. The pharmaceutical composition of claim 17, wherein the adjuvant is selected from the group consisting of mineral gels such as aluminum hydroxide, aluminum salts (e.g., aluminum phosphate) or calcium salts (e.g., calcium phosphate); MF59, or SAF; adjuvant systems (AS01, AS02, AS03, AS04) (GlaxoSmithKline), complete Freund's adjuvant, incomplete Freund's adjuvant, microbially-derived adjuvants such as cholera toxin (CT), pertussis toxin, Escherichia coli heat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72), Bacille Calmette-Guerin (BCG), lipopolysaccharides (LPS), mycobacteria, tetanus toxin, Corynebacterium parvum, DNA CpG motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate adjuvants such as immunostimulatory complexes (ISCOMs), liposomes, biodegradable microspheres, or saponins (e.g., QS-21); cytokines such as IFN-γ, IL-1, IL-2, IL-12 or GM-CSF; synthetic adjuvants such as nonionic block copolymers or surfactants, muramyl peptide analogues (e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine [thr-MDP], N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-[l'-2'-dipalmitoyl-s- n-glycero-3-hydroxyphosphoryloxy]- ethylamine), polyphosphazenes, synthetic polynucleotides, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, hydrocarbon emulsions, or keyhole limpet hemocyanins (KLH), CMC (carboxyl methylcellulose), HPMC (hydroxypropyl methylcellulose), glucopyranosyl Lipid adjuvant (GLA), or polylCLC.

19. A method to induce an immune response to HIV comprising: administering to a subject an effective amount of the recombinant MVA vector of claim 1, the recombinant DNA vector of claim 12 or a combination thereof to prime an immune response, wherein the vector comprises a nucleic acid insert comprising an HIV gag sequence and sequence encoding an HIV Env, thereby inducing an immune response.

20. The method of claim 19, wherein the immune response comprises an antibody response.

21. The method of claim 19, wherein the method further comprises administering a protein boost selected from the group consisting of recombinant gpl20, gpl20deltaN, or gpl40 SOSIP trimer protein subunits to boost a primed immune response.

22. The method of claim 16, wherein the induced immune response comprises a broadly neutralizing antibody (bnAb) response.

23. The method of claim 20 or 22 wherein the antibodies are CD4bs antibodies.

24. The method of claim 22, wherein the neutralizing antibodies are autologous.

25. The method of claim 19, wherein both the recombinant DNA vector and the MVA vector are administered, wherein the recombinant DNA vector is administered as a prime and the recombinant MVA vector is administered as a boost.

26. The method of claim 25, wherein the recombinant DNA vector, the recombinant MVA vector or both is administered in the form of a pharmaceutical composition.

27. The method of claim 25, wherein the nucleic acid insert of the recombinant DNA encodes an HIV Env that binds with an UCA and the MVA vector boost expresses the same HIV Env.

28. The method of claim 25, wherein the nucleic acid insert of both the recombinant DNA vector and the recombinant MVA vector encodes an HIV Env displayed on VLPs that binds with an UCA for bnAb, wherein the HIV Env is the same expressed from both vectors.

29. The method of claim 25, wherein the nucleic acid insert of both the DNA vector and the MVA vector encodes CH0505 T/F Env, and wherein the CH0505 T/F Env is displayed on VLPs and binds with an UCA for bnAb.

30. The method of claim 25 wherein the DNA vector composition comprises the CH0505 T/F Env displayed on VLPs that binds with an UCA for bnAb and the MVA boost comprises a different Env, wherein the different HIV Env is displayed on VLPs.

31. The method of claim 25 wherein the DNA vector and the MVA vector encodes EB354 T/F Env, and wherein the EB354 Env is displayed on VLPs that binds with an UCA for bnAb.

32. The method of claim 25 wherein the DNA vector composition comprises the EB354 T/F Env displayed on VLPs that binds with an UCA for bnAb and the MVA boost comprises a different Env, wherein the different HIV Env is displayed on VLPs.

33. The method of claim 25 wherein the composition further comprises a gpl20 protein, a gpl20deltaN, or gpl40 SOSIP trimer to enhance responses primed by the Envs displayed on VLPs by the DNA and MVA immunogenic compositions.

34. The method of claim 25 wherein the MVA vector is administered at least two times.

35. The method of claim 25 wherein the MVA vector is administered 2-4 times, 2-6 times, or 2-8 times.

36. The method of claim 25 wherein the DNA prime comprises CH0505 T/F HIV Env as gpl60 or gpl50, the MVA vector boost comprises CH0505 T/F HIV Env as gpl50 or gpl40, and wherein the method further comprises administering a boost with a recombinant MVA that expresses gpl50 of CH0505 T/F HIV Env, week 53.16 Env, week 78.33 HIV Env, or week 100.B6 HIV Env, and wherein the method further comprises administering a boost with a recombinant gpl20 HIV Env, gpl20deltaN or gpl40 SOSIP trimer HIV Env for the CH0505 T/F HIV Env, week 53.16 HIV Env, week 78.33 HIV Env, or week 100.B6 Envs.

37. The method of claim 25 wherein the DNA prime comprises a vector encoding EB354 T/F HIV Env, the MVA boost comprises a vector encoding EB354 T/F HIV Env, and wherein the method further comprises administering a boost with one or more of i) a recombinant gpl20 T/F HIV Env, ii) gpl20deltaN T/F HIV Env or iii) gpl40 SOSIP trimer HIV Env.

38. A method of broadening a broadly neutralizing antibody response to HIV from an unmutated common ancestor comprising: a) administering a composition to prime an immune response comprising a DNA vector of claim 12 expressing VLPs displaying one or more HIV Env, wherein the one or more Env are capable of interacting with unmutated common ancestors (UCAs) for broadly

neutralizing antibody (nAb) to initiate the Ab response, and b) administering a composition to boost a primed immune response, comprising a recombinant MVA vector of claim 1 expressing VLPs displaying the same or different one or more HIV Envs as the DNA vector composition, and c) administering one or more immunogenic vectors expressing mutated forms of the HIV Env protein that co-evolved with the generation of bnAb to broaden the primed immune response of a).

39. The method of claim 38 wherein the boosting vectors express VLPs displaying Env sequences selected from CH0505 T/F, week 53.16 Env, week 78.33 Env, and/or week

100.B6.

40. The method of claim 38 wherein the boosting vectors express VLPs displaying Env sequences selected from EB354 T/F, EB354 2010 Env, EB354 2014 Env, and/or EB354 2015 Env.

41. A kit comprising a DNA prime-MVA boost immunogenic composition comprising the MVA vector of claim 1 and the DNA vector of claim 12.