SWARM IMMUNIZATION WITH ENVELOPES FROM CH505

[0001] The application claims the benefit and priority of U.S. Serial No. 62/149,995 filed April 20, 2015, the content of which application is herein incorporated by reference in its entirety.

[0002] This invention was made with government support under Center for HIV/ AIDS Vaccine Immunology-Immunogen Design grant UM1-AI100645 from the NIH, NIAID, Division of AIDS. The government has certain rights in the invention.

[0003] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.

TECHNICAL FIELD

[0004] The present invention relates in general, to a composition suitable for use in inducing anti- HIV-1 antibodies, and, in particular, to immunogenic compositions comprising envelope proteins and nucleic acids to induce cross-reactive neutralizing antibodies and increase their breadth of coverage. The invention also relates to methods of inducing such broadly neutralizing anti-HIV-1 antibodies using such compositions.

BACKGROUND

[0005] 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 not routinely available in developing countries.

SUMMARY OF THE INVENTION

[0006] In certain embodiments, the invention provides compositions and method for induction of immune response, for example cross-reactive (broadly) neutralizing Ab induction. In certain embodiments, the methods use compositions comprising "swarms" of sequentially evolved envelope viruses that occur in the setting of bnAb generation in vivo in HIV-1 infection.

[0007] In certain aspects the invention provides compositions comprising a selection of HIV-1 envelopes and/ or nucleic acids encoding these envelopes as described herein for example but not limited to Selections as described herein. Without limitations, these selected combinations comprise envelopes which provide representation of the sequence (genetic) and antigenic diversity of the HIV-1 envelope variants which lead to the induction and maturation of the CH103 and CH235 antibody lineages. In certain embodiments the selections of envelopes comprise envelopes which show differential binding to an antibody or antibodies from CHI 03 and CH235 lineages. In certain embodiments, these compositions are used in immunization methods as a prime and/or boost.

[0008] In one aspect the invention provides selections of envelopes from individual CH505, which selections can be used in compositions for immunizations to induce lineages of broad neutralizing antibodies. In certain embodiments, there is some variance in the immunization regimen; in some embodiments, the selection of HIV-1 envelopes may be grouped in various combinations of primes and boosts, either as nucleic acids, proteins, or combinations thereof. In certain embodiments the compositions are pharmaceutical compositions which are immunogenic. In certain embodiments, the compositions comprise amounts of envelopes which are therapeutic and/or immunogenic.

[0009] In one aspect the invention provides a composition comprising any one of the envelopes described herein, or any combination thereof (selections in Examples). In some embodiments, CH505 Ml 1 Env is administered first as a prime, followed by a mixture of a next group of Envs. In some embodiments Ml 1 and M5 are administered as prime. In some embodiments, the prime is M5. In some embodiments, grouping of the envelopes is based on their binding affinity for the antibodies expected to be induced. In some embodiments, grouping of the envelopes is based on chronological evolution of envelope viruses that occurs in the setting of bnAb generation in vivo in HIV-1 infection. In some embodiments Loop D mutants could be included in either prime and/or boost. In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition and methods comprise use of agents for transient modulation of the host immune response.

[0010] In one aspect the invention provides a composition comprising a nucleic acid encoding HIV-1 envelope from Table I, III, V or any combination thereof. In certain embodiments the envelopes bind preferentially to an antibody or antibodies from CHI 03 lineage. In certain embodiments the envelopes bind preferentially to an antibody or antibodies from CH235 lineage. In one aspect the invention provides a composition comprising an HIV-1 envelope polypeptide from Table I, III, V or any combination thereof. In certain embodiments the envelopes bind preferentially to an antibody or antibodies from CHI 03 lineage. In certain embodiments the envelopes bind preferentially to an antibody or antibodies from CH235 lineage. [0011] In one aspect the invention provides a composition comprising nucleic acids encoding HIV- 1 envelope which is a loop D mutant, e.g. Ml 1 or any other suitable D loop mutant or combination thereof, e.g. Ml 1 and M5.

[0012] In one aspect, the invention provides a composition comprising a nucleic acid encoding HIV-1 envelope Mi l, w020.14, w030.28, w078.15, wl00.B6, or any combination thereof. In another aspect, the invention provides a composition comprising a nucleic acid encoding HIV-1 envelope Ml 1, M5, w020.14, w030.28, w078.15, wl00.B6, or any combination thereof. In another aspect, the invention provides a composition comprising a nucleic acid encoding HIV-1 envelope Mi l, M5, w020.14, w030.28, w078.15, w053.16, w030.21, w078.33, wl00.B6, w053.31, or any combination thereof.

[0013] In one aspect, the invention provides a composition comprising an HIV-1 envelope polypeptide Mi l, w020.14, w030.28, w078.15, wl00.B6, or any combination thereof. In another aspect, the invention provides a composition comprising an HIV-1 envelope polypeptide Ml 1, M5, w020.14, w030.28, w078.15, wl00.B6, or any combination thereof. In another aspect, the invention provides a composition comprising an HIV-1 envelope polypeptide Mi l, M5, w020.14, w030.28, w078.15, w053.16, w030.21, w078.33, wl00.B6, w053.31, or any combination thereof.

[0014] In one aspect, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides Mi l; M5; w020.14; w030.28; w078.15, w053.31, or a nucleic acid encoding any one of the HIV-1 envelope polypeptides Mi l; M5; w020.14; w030.28; w078.15, w053.31, or a combination thereof. In another aspect, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides Mi l, w020.14, w030.28, w078.15, wl00.B6, or a nucleic acid encoding any one of the HIV-1 envelope polypeptides Ml 1, w020.14, w030.28, w078.15, wl00.B6, or a combination thereof. In another aspect, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides Mi l, M5, w020.14, w030.28, w078.15, wl00.B6, or a nucleic acid encoding any one of the HIV-1 envelope polypeptides Ml 1, M5, w020.14, w030.28, w078.15, wl00.B6, or a combination thereof. In another aspect, the invention provides a composition comprising any one of the HIV-1 envelope polypeptides Ml 1, M5, w020.14, w030.28, w078.15, w053.16, w030.21, w078.33, wl00.B6, w053.31, or a nucleic acid encoding any one of the HIV-1 envelope polypeptides Ml 1, M5, w020.14, w030.28, w078.15, w053.16, w030.21, w078.33, wl00.B6, w053.31, or a combination thereof.

[0015] In one aspect, the invention provides a composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1, M5, w020.14, w030.28, w078.15, and w053.31. In another aspect, the invention provides a composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1, w020.14, w030.28, w078.15, and wl00.B6. In another aspect, the invention provides a composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Mi l, M5, w020.14, w030.28, w078.15, and wl00.B6. In another aspect, the invention provides a composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1, M5, w020.14, w030.28, w078.15, W053.16, w030.21, w078.33, wl00.B6, w053.31.

[0016] In another aspect the invention provides a method of inducing an immune response in a subject comprising administering a composition comprising HIV-1 envelope Ml 1 and/or M5 as a prime in an amount sufficient to induce an immune response, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same. A method of inducing an immune response in a subject comprising administering a composition comprising HIV-1 envelope Ml 1 and M5 as a prime in an amount sufficient to induce an immune response, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same.

[0017] In certain embodiments the methods further comprise administering a composition comprising any one of HIV-1 envelope Mi l; w020.14; w030.28; w078.15; w053.31 or any combination thereof as a boost, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same.

[0018] In certain embodiments the methods comprise administering a composition comprising any one of HIV-1 envelope Mi l; M5; w020.14; w030.28; w078.15; w053.31 or any combination thereof as a boost, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same.

[0019] In another aspect the invention provides a method of inducing an immune response in a subject comprising administering a composition comprising HIV-1 envelope Ml 1; M5; w020.14; w030.28; w078.15; w053.16; w030.21; w078.33; wl00.B6; w053.31 or any combination thereof as a prime and/or boost in an amount sufficient to induce an immune response, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same.

[0020] In certain embodiments the methods comprise administering a composition comprising any one of HIV-1 envelope Mi l, w020.14, w030.28, w078.15, wl00.B6 or any combination thereof, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same. In certain embodiments the methods comprise administering a composition comprising any one of HIV-1 envelope Mi l, M5, w020.14, w030.28, w078.15, wl00.B6 or any combination thereof, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same. In certain embodiments the methods comprise administering a composition comprising any one of HIV-1 envelope Mi l, M5, w020.14, w030.28, w078.15, w053.16, w030.21, w078.33, wl00.B6, w053.31 or any combination thereof, wherein the envelope is administered as a polypeptide or a nucleic acid encoding the same. In some embodiment, the envelope is administered as a boost.

[0021] In certain embodiments the methods comprise administering a first composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1; administering a second composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w020.14; administering a third

composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w030.28 and w078.15; and administering a fourth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides, w053.31. In some embodiments, the first composition is administered as a prime. In some embodiments, the second, third, and fourth compositions are administered as a boost. In some embodiments, the compositions are administered sequentially, in any order. In some embodiments, the first composition is administered as a prime and the second, third and fourth compositions are administered sequentially, in any order, after the administration of the first composition. In some embodiments, the first composition is administered before the second composition, the second composition is administered before the third composition, and the third composition is administered before the fourth composition.

[0022] In certain embodiments the methods comprise administering a first composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1; administering a second composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides M5; administering a third composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w020.14; administering a fourth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w030.28 and w078.15; and administering a fifth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides wl00.B6. In some embodiments, the first composition is administered as a prime. In some embodiments, second composition is administered as a prime, a boost, or both. In some embodiments, the third, fourth, and fifth compositions are administered as a boost. In some embodiments, the compositions are administered sequentially, in any order. In some embodiments, the first composition is administered as a prime and the second, third, fourth, and fifth compositions are administered sequentially, in any order, after the administration of the first composition. In some embodiments, the first composition is administered before the second composition, the second composition is administered before the third composition, the third composition is administered before the fourth composition, and the fourth composition is administered before the fifth composition.

[0023] In certain embodiments the methods comprise administering a first composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1; administering a second composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides M5; administering a third composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w020.14; administering a fourth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w030.28 and w078.15; and administering a fifth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w053.31. In some embodiment, the first composition is administered as a prime. In some embodiments, second composition is administered as a prime, a boost, or both. In some embodiments, the third, fourth, and fifth compositions are administered as a boost. In some embodiments, the compositions are administered sequentially, in any order. In some embodiments, the first composition is administered as a prime and the second, third, fourth, and fifth compositions are administered sequentially, in any order, after the administration of the first composition. In some embodiments, the first composition is administered before the second composition, the second composition is administered before the third composition, the third composition is administered before the fourth composition, and the fourth composition is administered before the fifth composition.

[0024] In certain embodiments the methods comprise administering a first composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides Ml 1; administering a second composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides M5; administering a third composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w020.14; administering a fourth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w030.28 and w078.15; administering a fifth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w053.16; administering a sixth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w030.21; administering a seventh composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w078.33; administering an eighth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides wl00.B6; and administering a ninth composition comprising the HIV-1 envelope polypeptides and/or nucleic acids encoding the HIV-1 envelope polypeptides w053.31. In some embodiment, the first composition is administered as a prime. In some embodiments, second composition is administered as a prime, a boost, or both. In some embodiments, the third, fourth, fifth, sixth, seventh, and eighth compositions are administered as a boost. In some embodiments, the compositions are administered sequentially, in any order. In some embodiments, the first composition is administered as a prime and the second, third, fourth, fifth, sixth, seventh, and eighth compositions are administered sequentially, in any order, after the administration of the first composition. In some embodiments, the first composition is

administered before the second composition, the second composition is administered before the third composition, the third composition is administered before the fourth composition, the fourth composition is administered before the fifth composition, the fifth composition is administered before the sixth composition, the sixth composition is administered before the seventh composition, and the seventh composition is administered before the eighth composition.

[0025] In certain embodiments, the compositions contemplate nucleic acid, as DNA and/or RNA, or proteins immunogens either alone or in any combination. In certain embodiments, the methods contemplate genetic, as DNA and/or RNA, immunization either alone or in combination with envelope protein(s).

[0026] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted in an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0027] In certain embodiments the induced immune response includes induction of antibodies, including but not limited to autologous and/or cross-reactive (broadly) neutralizing antibodies against HIV-1 envelope. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are known in the art and are also described herein.

[0028] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector.

[0029] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0030] In certain aspects the invention provides a composition comprising at least one nucleic acid encoding HIV- 1 envelope Mi l; M5; w020.14; w030.28; w078.15; w053.16; w030.21; w078.33; wl00.B6; w053.31 or any combination thereof. Non-limiting examples of combinations are shown in Example 2.

[0031] In certain embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide instead of a nucleic acid sequence encoding the HIV-1 envelope. In certain

embodiments, the compositions and methods employ an HIV-1 envelope as polypeptide, a nucleic acid sequence encoding the HIV-1 envelope, or a combination thereof.

[0032] The envelope used in the compositions and methods of the invention can be a gpl60, gpl50, gpl45, gpl40, gpl20, gp41, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof.

[0033] The polypeptide contemplated by the invention can be a polypeptide comprising any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting essentially of any one of the polypeptides described herein. The polypeptide contemplated by the invention can be a polypeptide consisting of any one of the polypeptides described herein. In certain embodiments, the polypeptide is recombinantly produced. In certain embodiments, the polypeptides and nucleic acids of the invention are suitable for use as an immunogen, for example to be administered in a human subject.

[0034] In certain embodiments the envelope is any of the forms of HIV-1 envelope. In certain embodiments the envelope is gpl20, gpl40 , gpl45 (i.e. with a transmembrane). In certain embodiments, the envelope is in a liposome and transmembrane with a cytoplasmic tail in a liposome. In certain embodiments, the nucleic acid comprises a nucleic acid sequence which encode a gpl20, gpl40, gpl45, gpl50 or gpl60.

[0035] In certain embodiments, where the nucleic acids are operably linked to a promoter and inserted in a vector, the vectors is any suitable vector. Non-limiting examples, include, the VSV, replicating rAdenovirus type 4, MVA, Chimp adenovirus vectors, pox vectors, and the like. In certain embodiments, the nucleic acids are administered in NanoTaxi block polymer nanospheres.In certain embodiments, the composition and methods comprise an adjuvant. Non-limiting examples include, ASOl B, ASOl E, gla/SE, alum, Poly I poly C, TLR agonists, TLR7/8 and 9 agonists, or a combination of TLR7/8 and TLR9 agonists (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329-3339) , or any other adjuvant. Non-limiting examples of TLR7/8 agonist include TLR7/8 ligands, Gardiquimod, Imiquimod and R848 (resiquimod). A non-limiting embodiment of a combination of TLR7/8 and TLR9 agonist comprises R848 and oCpG in STS (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 6 3329-3339).

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color. In the below descriptions and the examples, the colored images are described in terms of its appearance in black and white. Different colors are described by different shades of white to gray with an attempt to match the description the descriptions of the color as closely as possible to that of the figures.

[0037] Figure 1 Shows sequences of six envelopes (SEQ. ID. NO.: 1-49), including sequences of 3' bar codes for various CH505 Env gpl45 and gpl20 (SEQ. ID. NO.: 1-6, in order of appearance in table); nucleotide sequences of: >HV1300656, CH505.M5gpl45 (SEQ. ID. NO. : 7);

>HV1300662, CH505.Mlgpl45 (SEQ. ID. NO. : 8); >HV1300635, CH505w020.14gpl45 (SEQ.

ID. NO.: 9); >HV1300636, CH505w030.28gpl45 (SEQ. ID. NO. : 10); >HV1300639,

CH505w078.15gpl45 (SEQ. ID. NO.: 11); >HV1300638, CH505w053.31gpl45 (SEQ. ID. NO.:

12); amino acid sequences of: >HV1300656, CH505.M5gpl45 (SEQ. ID. NO. : 13);

>HV1300662, CH505.M1 lgpl45 (SEQ. ID. NO.: 14); >HV1300635, CH505w020.14gpl45 (SEQ.

ID. NO.: 15); >HV1300636, CH505w020.14gpl45 (SEQ. ID. NO.: 16); >HV1300638,

CH505w053.31gpl45 (SEQ. ID. NO.: 17); >HV1300639, CH505w078.15gpl45 (SEQ. ID. NO.:

18); >HV1300638, CH505w053.31gpl45 (SEQ. ID. NO. : 19); sequences of bar codes for various

CH505 Env gpl20 (SEQ. ID. NO.: 20-25, in order of appearance in table); nucleotide sequences of:

>HV1300531_v2, CH505.M5D8gpl20 (SEQ. ID. NO.: 26); >HV1300537_v2,

CH505.Ml lD8gpl20 (SEQ. ID. NO. : 27); >HV1300556_v2, CH505w020.14D8gpl20 (SEQ. ID.

NO. : 28); >HV1300578_v2, CH505w030.28D8gpl20 (SEQ. ID. NO. : 29); >HV1300592,

CH505w078.15D8gpl20 (SEQ. ID. NO.: 30); >HV1300586, CH505w053.31D8gpl20 (SEQ. ID.

NO. : 31); amino acid sequences of: >HV1300531_v2, CH505.M5D8gpl20 (SEQ. ID. NO.: 32);

>HV1300537_v2, CH505.M1 lD8gpl20 (SEQ. ID. NO.: 33); >HV1300556_v2,

CH505w020.14D8gpl20 (SEQ. ID. NO.: 34); >HV1300578_v2, CH505w030.28D8gpl20 (SEQ. ID. NO.: 35); >HV1300592, CH505w078.15D8gpl20 (SEQ. ID. NO. : 36); >HV1300586,

CH505w053.31D8gpl20 (SEQ. ID. NO.: 37); nucleotide sequences of gpl20 constructs:

>CH505.M5gpl60 (SEQ. ID. NO. : 38); >CH505.M1 lgpl60 (SEQ. ID. NO. : 39);

>CH505w020.14gpl60 (SEQ. ID. NO. : 40); >CH505w030.28gpl60 (SEQ. ID. NO. : 41);

>CH505w078.15gpl60 (SEQ. ID. NO. : 42); >CH505w053.31gpl60 (SEQ. ID. NO. : 43); amino acid sequences of: >CH505.M5gpl60 (SEQ. ID. NO.: 44); >CH505.M1 lgpl60 (SEQ. ID. NO. : 45); >CH505w020.14gpl60 (SEQ. ID. NO.: 46); >CH505w030.28gpl60 (SEQ. ID. NO. : 47); >CH505w078.15gpl60 (SEQ. ID. NO. : 48); >CH505w053.31gpl60 (SEQ. ID. NO.: 49).

[0038] Figure 2 Shows sequences of ten envelopes (SEQ. ID. NO. : 50-126), including sequences of bar codes for various CH505 Env gpl45 (SEQ. ID. NO.: 50-59, in order of appearance in table); nucleotide sequences of: >HV1300656, CH505.M5gpl45 (SEQ. ID. NO.: 60); >HV1300662, CH505.Mlgpl45 (SEQ. ID. NO. : 61); >HV1300635, CH505w020.14gpl45 (SEQ. ID. NO. : 62); >HV1300636, CH505w030.28gpl45 (SEQ. ID. NO.: 63); >HV1300639, CH505w078.15gpl45 (SEQ. ID. NO. : 64); >HV1300696, CH505w53.16gpl45 (SEQ. ID. NO.: 65); >HV1300689, CH505w30.21gpl45 (SEQ. ID. NO.: 66); >HV1300705, CH505w78.33gpl45 (SEQ. ID. NO.: 67); >HV1300714, CH505wl00.B6gpl45 (SEQ. ID. NO. : 68); >HV1300638, CH505w053.31gpl45 (SEQ. ID. NO. : 69); amino acid sequences of: >HV1300656, CH505.M5gpl45 (SEQ. ID. NO.: 70); >HV1300662, CH505.M1 lgpl45 (SEQ. ID. NO. : 71); >HV1300635, CH505w020.14gpl45 (SEQ. ID. NO. : 72); >HV1300636, CH505w020.14gpl45 (SEQ. ID. NO.: 73); >HV1300638, CH505w053.31gpl45 (SEQ. ID. NO.: 74); >HV1300696, CH505w53.16gpl45 (SEQ. ID. NO. : 75); >HV1300689, CH505w30.21gpl45 (SEQ. ID. NO. : 76); >HV1300705, CH505w78.33gpl45 (SEQ. ID. NO. : 77); >HV1300714, CH505wl00.B6gpl45 (SEQ. ID. NO.: 78); >HV1300639, CH505w078.15gpl45 (SEQ. ID. NO.: 79); >HV1300638, CH505w053.31gpl45 (SEQ. ID. NO. : 80); sequences of bar codes for various CH505 Env gpl20 (SEQ. ID. NO. : 81-86, in order of appearance in table); nucleotide sequences of: >HV1300531_v2, CH505.M5D8gpl20 (SEQ. ID. NO. : 87); >HV1300537_v2, CH505.M1 lD8gpl20 (SEQ. ID. NO.: 88); >HV1300556_v2,

CH505w020.14D8gpl20 (SEQ. ID. NO.: 89); >HV1300578_v2, CH505w030.28D8gpl20 (SEQ. ID. NO.: 90); >HV1300592, CH505w078.15D8gpl20 (SEQ. ID. NO. : 91); >HV1300583,

CH505w053.16D8gpl20 (SEQ. ID. NO.: 92); >HV1300574_v2, CH505w030.21D8gpl20 (SEQ. ID. NO.: 93); >HV1300595, CH505w078.33D8gpl20 (SEQ. ID. NO. : 94); >HV1300605,

CH505wl00.B6D8gpl20 (SEQ. ID. NO.: 95); >HV1300586, CH505w053.31D8gpl20 (SEQ. ID. NO. : 96); ); amino acid sequences of: >HV1300531_v2, CH505.M5D8gpl20 (SEQ. ID. NO. : 97); >HV1300537_v2, CH505.M1 lD8gpl20 (SEQ. ID. NO.: 98); >HV1300556_v2,

CH505w020.14D8gpl20 (SEQ. ID. NO.: 99); >HV1300578_v2, CH505w030.28D8gpl20 (SEQ. ID. NO.: 100); >HV1300592, CH505w078.15D8gpl20 (SEQ. ID. NO. : 101); >HV1300583, CH505w053.16D8gpl20 (SEQ. ID. NO. : 102); >HV1300574_v2, CH505w030.21D8gpl20 (SEQ. ID. NO.: 103); >HV1300595, CH505w078.33D8gpl20 (SEQ. ID. NO. : 104); >HV1300605, CH505wl00.B6D8gpl20 (SEQ. ID. NO.: 105); >HV1300586, CH505w053.31D8gpl20 (SEQ. ID. NO. : 106); nucleotide sequences of gpl60 constructs: >CH505.M5gpl60 (SEQ. ID. NO.: 107); >CH505.Ml lgpl60 (SEQ. ID. NO. : 108); >CH505w020.14gpl60 (SEQ. ID. NO. : 109);

>CH505w030.28gpl60 (SEQ. ID. NO. : 110); >CH505w078.15gpl60 (SEQ. ID. NO.: I l l);

>CH505w053.16gpl60 (SEQ. ID. NO. : 112); >CH505w030.21gpl60 (SEQ. ID. NO.: 113);

>CH505w078.33gpl60 (SEQ. ID. NO. : 114); >CH505wl00.B6gpl60 (SEQ. ID. NO.: 115);

>CH505w053.31gpl60 (SEQ. ID. NO. : 116); amio acid sequences of: >CH505.M5gpl60 (SEQ. ID. NO.: 117); >CH505.M1 lgpl60 (SEQ. ID. NO. : 118); >CH505w020.14gpl60 (SEQ. ID. NO.: 119); >CH505w030.28gpl60 (SEQ. ID. NO.: 120); >CH505w078.15gpl60 (SEQ. ID. NO. : 121); >CH505w053.16gpl60 (SEQ. ID. NO. : 122); >CH505w030.21gpl60 (SEQ. ID. NO.: 123);

>CH505w078.33gpl60 (EQ. ID. NO. : 124); >CH505wl00.B6gpl60 (SEQ. ID. NO. : 125);

>CH505w053.31gpl60 (SEQ. ID. NO.: 126).

[0039] Figures 3 A-3C show the genotype variation (Fig. 3 A), neutralization titers (Fig. 3B), and Envelope phylogenetic relations (Fig. 3C) among CH505 Envelope variants. The vertical position in each panel corresponds to the same CH505 Env clone named on the right side of the tree.

Distance from the Transmitted/Founder form generally increases from top towards bottom of the figure. In Fig. 3 A, sites not colored correspond to the Transmitted/Founder virus, dark grey sites show mutations, and black sites correspond to insertions or deletions relative to the

Transmitted/Founder virus. Additional annotation indicates the known CD4 binding-site contacts (short, vertical black bars towards top), CHI 03 binding-site contacts for the resolved structure (short, vertical grey bars with a horizontal line to indicate the region resolved by X-Ray

Crystallography), gpl20 landmarks (vertical grey rectangular regions, V1-V5 hypervariable loops, Loop D, and CD4 Loops), a dashed vertical line delineating the gpl20/gp41 boundary, and results from testing for CTL epitopes with ELISpot assays (light grey horizontal bands at top and bottom show where peptides were tested and negative, and a light grey rectangle for the tested positive region outside the C-terminal end of V4). Fig. 3B depicts IC50 (50% inhibitory concentrations, in μg/ml) values from autologous neutralization assays against 13 monoclonal antibodies (MAbs) of the CHI 03 lineage and each of 134 CH505 Env-pseudotyped viruses. Grey color-scale values indicate neutralization potency and range from grey (no neutralization detected) through dark grey (potent neutralization, i.e. <0.2 μg/ml; empty cells correspond to absence of information). The cumulative progression of neutralization potency from left to right, corresponding to developmental stages in the CHI 03 lineage, indicates accumulation of neutralization potency. Similarly, increased presence neutralization signal from top to bottom corresponds to increasing neutralization breadth per MAb in the CH103 lineage. In Fig. 3C) is the phylogeny of CH505 Envs, with the x-axis indicating distance from the Transmitted-Founder virus per the scale bar (units are mutations per site). The tree is ordered vertically such that lineages with the most descendants appear towards the bottom. Each leaf on the tree corresponds to a CH505 autologous Env, with the name of the sequence depicted ('w' and symbol color indicate the sample time-point; 'M' indicates a synthetic mutant Env). The color of text in each leaf name indicates its inclusion in a possible embodiment. Three long, vertical lines to the left of the tree depict the phylogenetic distribution of envelopes in three distinct alternative embodiments (identified as "Vaccination Regimes 1-3"), with diamonds used to identify each.

[0040] Figures 4-8 show Heat Map of Binding (log Area Under the Curve, AUC) of Sequential Envs to CHI 03 and CH235 CD4 Binding Site Broadly Neutralizing Antibody Lineages members. Numerical data corresponding to the graphic representations in these figures are shown in Tables 1- 4.

[0041] Figure 9 shows neutralization activity of CHI 03 clonal lineage antibodies against autologous CH505 viruses at various time points (week 4, week 14, week 20, week 30, week 53, week 78, and week 100). Values are the concentrations ^g/mL) of antibodies required for the 50% inhibition (IC ₅₀ ).

[0042] Figure 10 shows neutralization susceptibility of the CH505 loop D mutants to CHI 03 lineage antibodies. Values are the concentrations ^g/mL) of antibodies required for the 50% inhibition (IC ₅₀ ).

[0043] Figure 11 shows the binding data and choice of immunogens for CHI 03.

[0044] Figure 12 shows neutralization susceptibility of the CH505 loop D mutants to CH235 lineage antibodies. Values are the concentrations ^g/mL) of antibodies required for the 50% inhibition (IC ₅₀ ).

[0045] Figure 13 shows neutralization activity of CH235 clonal lineage antibodies against autologous CH505 viruses at various time points (week 4, week 14, week 20, week 30, week 53, week 78, and week 100). Values are the concentrations ^g/mL) of antibodies required for the 50% inhibition (IC ₅₀ ).

DETAILED DESCRIPTION OF THE INVENTION

[0046] 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: 225- 244, 2013). BnAbs are protective in rhesus macaques against SHIV challenge, but as yet, are not induced by current vaccines.

[0047] 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.

[0048] Recently, a new paradigm for design of strategies for induction of broadly neutralizing antibodies was introduced, that of B cell lineage immunogen design (Nature Biotech. 30: 423, 2012) in which the induction of bnAb lineages is recreated. It was recently demonstrated the power of mapping the co-evolution of bnAbs and founder virus for elucidating the Env evolution pathways that lead to bnAb induction (Nature 496: 469, 2013). From this type of work has come the hypothesis that bnAb induction will require a selection of antigens to recreate the "swarms" of sequentially evolved viruses that occur in the setting of bnAb generation in vivo in HIV infection (Nature 496: 469, 2013).

[0049] A critical question is why the CH505 immunogens are better than other immunogens. This rationale comes from three recent observations. First, a series of immunizations of single putatively "optimized" or "native" trimers when used as an immunogen have not induced bnAbs as single immunogens. Second, in all the chronically infected individuals who do develop bnAbs, they develop them in plasma after ~2 years. When these individuals have been studied at the time soon after transmission, they do not make bnAbs immediately. Third, now that individual's virus and bnAb co-evolution has been mapped from the time of transmission to the development of bnAbs, the identification of the specific Envs that lead to bnAb development have been identified- thus taking the guess work out of envelope choice.

[0050] Two other considerations are important. The first is that for the CHI 03 bnAb CD4 binding site lineage, the VH4-59 and νλ3-1 genes are common as are the VDJ, VJ recombinations of the lineage (Liao, Nature 496: 469, 2013). In addition, the bnAb sites are so unusual, we are finding that the same VH and VL usage is recurring in multiple individuals. Thus, we can expect the CH505 Envs to induce CD4 binding site antibodies in many different individuals.

[0051] Finally, regarding the choice of gpl20 vs. gpl60, for the genetic immunization we would normally not even consider not using gpl60. However, in acute infection, gp41 non-neutralizing antibodies are dominant and overwhelm gpl20 responses (Tomaras, G et al. J. Virol. 82: 12449, 2008; Liao, HX et al. JEM 208: 2237, 2011). Recently we have found that the HVTN 505 DNA prime, rAd5 vaccine trial that utilized gpl40 as an immunogen, also had the dominant response of non-neutralizing gp41 antibodies. Thus, we will evaluate early on the use of gpl60 vs gpl20 for gp41 dominance.

[0052] In certain aspects the invention provides a strategy for induction of bnAbs is to select and develop immunogens and combinations designed to recreate the antigenic evolution of Envs that occur when bnAbs do develop in the context of infection.

[0053] That broadly neutralizing antibodies (bnAbs) occur in nearly all sera from chronically infected HIV-1 subjects suggests anyone can develop some bnAb response if exposed to immunogens via vaccination. Working back from mature bnAbs through intermediates enabled understanding their development from the unmutated ancestor, and showed that antigenic diversity preceded the development of population breadth. See Liao et al. (2013) Nature 496, 469-476. In this study, an individual "CH505" was followed from HIV-1 transmission to development of broadly neutralizing antibodies. This individual developed antibodies targeted to CD4 binding site on gpl20. In this individual the virus was sequenced over time, and broadly neutralizing antibody clonal lineage ("CHI 03") was isolated by antigen-specific B cell sorts, memory B cell culture, and amplified by VH/VL next generation pyrosequencing. The CHI 03 lineage began by binding the T/F virus, autologous neutralization evolved through somatic mutation and affinity maturation, escape from neutralization drove rapid (clearly by 20 weeks) accumulation of variation in the epitope, antibody breadth followed this viral diversification.

[0054] Further analysis of envelopes and antibodies from the CH505 individual indicated that a non-CH103 Lineage (DH235) participates in driving CH103-BnAb induction. See Gao et al. (2014) Cell 158:481-491. For example VI loop, V5 loop and CD4 binding site loop mutations escape from CHI 03 and are driven by CHI 03 lineage. Loop D mutations enhanced neutralization by CHI 03 lineage and are driven by another lineage. Transmitted/founder Env, or another early envelope for example W004.26, triggers naive B cell with CHI 03 Unmutated Common Ancestor (UCA) which develop in to intermediate antibodies. Transmitted/founder Env, or another early envelope for example W004.26, also triggers non-CHI 03 autologous neutralizing Abs that drive loop D mutations in Env that have enhanced binding to intermediate and mature CHI 03 antibodies and drive remainder of the lineage. In certain embodiments, the inventive composition and methods also comprise loop D mutant envelopes (e.g. but not limited to M10, Mi l, M19, M20, M21, M5, M6, M7, M8, M9) as immunogens. In certain embodiments, the D-loop mutants are included in an inventive composition used to induce an immune response in a subject. In certain embodiments, the D-loop mutants are included in a composition used as a prime.

[0055] The invention provides various methods to choose a subset of viral variants, including but not limited to envelopes, to investigate the role of antigenic diversity in serial samples. In other aspects, the invention provides compositions comprising viral variants, for example but not limited to envelopes, selected based on various criteria as described herein to be used as immunogens. In some embodiments, the immunogens are selected based on the envelope binding to the UCA, and/or intermediate antibodies. In some embodiments the immunogens are selected based on their chronological appearance and/or sequence diversity during infection.

[0056] In other aspects, the invention provides immunization strategies using the selections of immunogens to induce cross-reactive neutralizing antibodies. In certain aspects, the immunization strategies as described herein are referred to as "swarm" immunizations to reflect that multiple envelopes are used to induce immune responses. The multiple envelopes in a swarm could be combined in various immunization protocols of priming and boosting.

[0057] In certain embodiments the invention provides that sites losing the ancestral, transmitted- founder (T/F) state are most likely under positive selection. From acute, homogenous infections with 3-5 years of follow-up, identified herein are sites of interest among plasma single genome analysis (SGA) Envs by comparing the proportion of sequences per time-point in the T/F state with a threshold, typically 5%. Sites with T/F frequencies below threshold are putative escapes. We then selected clones with representative escape mutations. Where more information was available, such as tree-corrected neutralization signatures and antibody contacts from co-crystal structure, additional sites of interest were considered.

[0058] Co-evolution of a broadly neutralizing HIV-1 antibody (CHI 03) and founder virus was previously reported in African donor (CH505). See Liao et al. (2013) Nature 496, 469-476. In CH505, which had an early antibody that bound autologous T/F virus, we studied 398 envs from 14 time-points over three years (median per sample: 25, range: 18-53). We found 36 sites with T/F frequencies under 20% in any sample. Neutralization and structure data identified 28 and 22 interesting sites, respectively. Together, six gp41 and 53 gpl20 sites were identified, plus six VI or V5 insertions not in HXB2.

[0059] The invention provides an approach to select reagents for neutralization assays and subsequently investigate affinity maturation, autologous neutralization, and the transition to heterologous neutralization and breadth. Given the sustained coevolution of immunity and escape this antigen selection based on antibody and antigen coevolution has specific implications for selection of immunogens for vaccine design.

[0060] In one embodiment, five envelopes were selected that represent envelope antigenic diversity. In another embodiment, six envelopes were selected that represent envelope antigenic diversity. In another embodiment, ten envelopes were selected that represent envelope antigenic diversity. These sets of envelopes represent antigenic diversity by deliberate inclusion of polymorphisms that result from immune selection by neutralizing antibodies. These selections represent various levels of antigenic diversity in the HIV-1 envelope. In some embodiments the selections are based on the genetic diversity of longitudinally sampled SGA envelopes. In some embodiments the selections are based on antigenic and or neutralization diversity. In some embodiments the selections are based on the genetic diversity of longitudinally sampled SGA envelopes, and correlated with other factors such as antigenic/neutralization diversity, and antibody coevolution.

[0061] Sequences/Clones

[0062] Described herein are nucleic and amino acids sequences of HIV-1 envelopes. In certain embodiments, the described HIV-1 envelope sequences are gpl60s. In certain embodiments, the described HIV-1 envelope sequences are gpl20s. Other sequences, for example but not limited to gpl45s, gpl40s, both cleaved and uncleaved, gpl 50s, gp41 s, which are readily derived from the nucleic acid and amino acid gpl 60 sequences. In certain embodiments the nucleic acid sequences are codon optimized for optimal expression in a host cell, for example a mammalian cell, a rBCG cell or any other suitable expression system.

[0063] In certain embodiments, the envelope design in accordance with the present invention involves deletion of residues (e.g., 5-1 1, 5, 6, 7, 8, 9, 10, or 1 1 amino acids) at the N-terminus. For delta N-terminal design, amino acid residues ranging from 4 residues or even fewer to 14 residues or even more are deleted. These residues are between the maturation (signal peptide, usually ending with CX, X can be any amino acid) and "VPVXXXX. . . ". In case of CH505 T/F Env as an example, 8 amino acids (italicized and underlined in the below sequence) were deleted:

M R V M G I Q R N Y P Q W W I W S M L G F W M L M I C N GA / WVl Ί ΎΥ(ί VPVWKEAKTTLF CASPAR AY EKEVHN VW ATH AC VPTDPNPQE . . . (rest of envelope sequence is indicated as " . . . "). In other embodiments, the delta N-design described for CH505 T/F envelope can be used to make delta N- designs of other CH505 envelopes. In certain embodiments, the invention relates generally to an immunogen, gpl60, gpl20 or gpl40, without an N-terminal Herpes Simplex gD tag substituted for amino acids of the N-terminus of gpl20, with an HIV leader sequence (or other leader sequence), and without the original about 4 to about 25, for example 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids of the N-terminus of the envelope (e.g. gpl20). See WO2013/006688, e.g. at pages 10-12, the contents of which publication is hereby incorporated by reference in its entirety.

[0064] The general strategy of deletion of N-terminal amino acids of envelopes results in proteins, for example gpl20s, expressed in mammalian cells that are primarily monomeric, as opposed to dimeric, and, therefore, solves the production and scalability problem of commercial gpl20 Env vaccine production. In other embodiments, the amino acid deletions at the N-terminus result in increased immunogenicity of the envelopes.

[0065] In certain embodiments, the invention provides envelope sequences, amino acid sequences and the corresponding nucleic acids, and in which the V3 loop is substituted with the following V3 loop sequence TRPNNNTRKSIRIGPGQTFY ATGDIIGNIRQAH (SEQ. ID. NO.: 127). This substitution of the V3 loop reduced product cleavage and improves protein yield during

recombinant protein production in CHO cells.

[0066] In certain embodiments, the CH505 envelopes will have added certain amino acids to enhance binding of various broad neutralizing antibodies. Such modifications could include but not limited to, mutations at W680G or modification of glycan sites for enhanced neutralization.

[0067] In certain aspects, the invention provides composition and methods which use a selection of sequential CH505 Envs, as gpl20s, gp 140s cleaved and uncleaved, gpl45s, gpl50s and gpl60s, as proteins, DNAs, RNAs, or any combination thereof, administered as primes and boosts to elicit immune response. Sequential CH505 Envs as proteins would be co-administered with nucleic acid vectors containing Envs to amplify antibody induction. In certain embodiments, the compositions and methods include any immunogenic HIV-1 sequences to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic and/or consensus HIV-1 genes to give the best coverage for T cell help and cytotoxic T cell induction. In certain embodiments, the compositions and methods include mosaic group M and/or consensus genes to give the best coverage for T cell help and cytotoxic T cell induction. In some embodiments, the mosaic genes are any suitable gene from the HIV-1 genome. In some embodiments, the mosaic genes are Env genes, Gag genes, Pol genes, Nef genes, or any combination thereof. See e.g. US Patent No. 7951377. In some embodiments the mosaic genes are bivalent mosaics. In some embodiments the mosaic genes are trivalent. In some embodiments, the mosaic genes are administered in a suitable vector with each immunization with Env gene inserts in a suitable vector and/or as a protein. In some embodiments, the mosaic genes, for example as bivalent mosaic Gag group M consensus genes, are administered in a suitable vector, for example but not limited to HSV2, would be administered with each immunization with Env gene inserts in a suitable vector, for example but not limited to HSV-2.

[0068] In certain aspects the invention provides compositions and methods of Env genetic immunization either alone or with Env proteins to recreate the swarms of evolved viruses that have led to bnAb induction. Nucleotide-based vaccines offer a flexible vector format to immunize against virtually any protein antigen. Currently, two types of genetic vaccination are available for testing— DNAs and mRNAs. [0069] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA. See Graham BS, Enama ME, Nason MC, Gordon IJ, Peel SA, et al. (2013) DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies for delivery of nucleic acids, as DNA and/or RNA, so as to elicit immune response, both T-cell and humoral responses, are known in the art and are under developments. In certain embodiments, DNA can be delivered as naked DNA. In certain embodiments, DNA is formulated for delivery by a gene gun. In certain embodiments, DNA is administered by electroporation, or by a needle-free injection technologies, for example but not limited to Biojector® device. In certain embodiments, the DNA is inserted in vectors. The DNA is delivered using a suitable vector for expression in mammalian cells. In certain

embodiments the nucleic acids encoding the envelopes are optimized for expression. In certain embodiments DNA is optimized, e.g. codon optimized, for expression. In certain embodiments the nucleic acids are optimized for expression in vectors and/or in mammalian cells. In non-limiting embodiments these are bacterially derived vectors, adenovirus based vectors, rAdenovirus (e.g. Barouch DH, et al. Nature Med. 16: 319-23, 2010), recombinant mycobacteria (e.g. rBCG or M smegmatis) (Yu, JS et al. Clinical Vaccine Immunol. 14: 886-093,2007; ibid 13 : 1204-11,2006), and recombinant vaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010), for example but not limited to ALVAC, replicating (Kibler KV et al., PLoS One 6: e25674, 2011 nov 9.) and non- replicating (Perreau M et al. J. virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MVA)), adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons, Herpes Simplex Virus vectors, and other suitable vectors.

[0070] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as DNA or RNA in suitable formulations. Various technologies which contemplate using DNA or RNA, or may use complexes of nucleic acid molecules and other entities to be used in immunization. In certain embodiments, DNA or RNA is administered as nanoparticles consisting of low dose antigen-encoding DNA formulated with a block copolymer (amphiphilic block copolymer 704). See Cany et al., Journal of Hepatology 2011 vol. 54 j 115- 121; Arnaoty et al., Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology, vol. 859, pp293-305 (2012); Arnaoty et al. (2013) Mol Genet Genomics. 2013 Aug;288(7-8):347-63. Nanocarrier technologies called

Nanotaxi® for immunogenic macromolecules (DNA, RNA, Protein) delivery are under

development. See for example technologies developed by incellart. [0071] In certain aspects the invention contemplates using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins suitable for use in immunization are known in the art. In certain embodiments recombinant proteins are produced in CHO cells.

[0072] The immunogenic envelopes can also be administered as a protein boost in combination with a variety of nucleic acid envelope primes (e.g., HIV -1 Envs delivered as DNA expressed in viral or bacterial vectors).

[0073] Dosing of proteins and nucleic acids can be readily determined by a skilled artisan. A single dose of nucleic acid can range from a few nanograms (ng) to a few micrograms ^g) or milligram 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.

[0074] Administration: 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 delivered via intramascular (IM), via subcutaneous, via intravenous, via nasal, via mucosal routes, or any other suitable route of immunization.

[0075] 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, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen [Leroux-Roels et al., IABS Conference, April 2013,9]. In certain embodiments, TLR agonists are used as adjuvants. In other embodiment, adjuvants which break immune tolerance are included in the immunogenic compositions.

[0076] In certain embodiments, the compositions and methods comprise any suitable agent or immune modulation which could modulate mechanisms of host immune tolerance and release of the induced antibodies. In non-limiting embodiments modulation includes PD-1 blockade; T regulatory cell depletion; CD40L hyperstimulation; soluble antigen administration, wherein the soluble antigen is designed such that the soluble agent eliminates B cells targeting dominant epitopes, or a combination thereof. In certain embodiments, an immunomodulatory agent is administered in at time and in an amount sufficient for transient modulation of the subject's immune response so as to induce an immune response which comprises broad neutralizing antibodies against HIV-1 envelope. Non-limiting examples of such agents is any one of the agents described herein: e.g. chloroquine (CQ), PTP1B Inhibitor - CAS 765317-72-4 - Calbiochem or MSI 1436 clodronate or any other bisphosphonate; a Foxol inhibitor, e.g. 344355 | Foxol Inhibitor, AS 1842856 - Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, an agent which binds to a B cell receptor for a dominant HIV-1 envelope epitope, or any combination thereof. In certain embodiments, the methods comprise administering a second immunomodulatory agent, wherein the second and first immunomodulatory agents are different.

[0077] There are various host mechanisms that control bNAbs. For example highly somatically mutated antibodies become autoreactive and/or less fit (Immunity 8: 751, 1998; PloS Comp. Biol. 6 el000800 , 2010; J. Thoret. Biol. 164:37, 1993); Polyreactive/autoreactive naive B cell receptors (unmutated common ancestors of clonal lineages) can lead to deletion of Ab precursors (Nature 373 : 252, 1995; PNAS 107: 181, 2010; J. Immunol. 187: 3785, 2011); Abs with long HCDR3 can be limited by tolerance deletion (JI 162: 6060, 1999; JCI 108: 879, 2001). BnAb knock-in mouse models are providing insights into the various mechanisms of tolerance control of MPER BnAb induction (deletion, anergy, receptor editing). Other variations of tolerance control likely will be operative in limiting BnAbs with long HCDR3s, high levels of somatic hypermutations.

[0078] The invention is described in the following non-limiting examples.

EXAMPLES

Example 1

[0079] HIV-1 sequences, including envelopes, and antibodies from HIV-1 infected individual CH505 were isolated as described in Liao et al. (2013) Nature 496, 469-476 including

supplementary materials; See also Gao et al. (2014) Cell 158:481-491.

[0080] Recombinant HIV-1 proteins

[0081] HIV-1 Env genes for subtype B, 63521, subtype C, 1086, and subtype CRF 01, 427299, as well as subtype C, CH505 autologous transmitted/founder Env were obtained from acutely infected HIV-1 subjects by single genome amplification, codon-optimized by using the codon usage of highly expressed human housekeeping genes, de novo synthesized (GeneScript) as 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. Select Env proteins made as CH505 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.

[0082] ELISA

[0083] Binding of patient plasma antibodies and CH103, and DH235(CH235), See Gao et al. (2014) Cell 158:481-491, clonal lineage antibodies to autologous and heterologous HIV-1 Env proteins was measured by ELISA as described previously. Plasma samples in serial threefold dilutions starting at 1 :30 to 1 :521,4470 or purified monoclonal antibodies in serial threefold dilutions starting at 100 μg ml-1 to 0.000 μg ml-1 diluted in PBS were assayed for binding to autologous and heterologous HIV-1 Env proteins. Binding of biotin-labelled CHI 03 at the subsaturating concentration was assayed for cross-competition by unlabeled HIV-1 antibodies and soluble CD4-Ig in serial fourfold dilutions starting at 10 μg ml-1. The half-maximal effective concentration (EC50) of plasma samples and monoclonal antibodies to HIV-1 Env proteins were determined and expressed as either the reciprocal dilution of the plasma samples or concentration of monoclonal antibodies.

[0084] Surface plasmon resonance affinity and kinetics measurements

[0085] Binding Kd and rate constant (association rate (Ka)) measurements of monoclonal antibodies and all candidate UCAs to the autologous Env C. CH05 gpl40 and/or the heterologous Env B.63521 gpl20 are carried out on BIAcore 3000 instruments as described previously. Anti- human IgG Fc antibody (Sigma Chemicals) is immobilized on a CM5 sensor chip to about 15,000 response units and each antibody is captured to about 50-200 response units on three individual flow cells for replicate analysis, in addition to having one flow cell captured with the control Synagis (anti-RSV) monoclonal antibody on the same sensor chip. Double referencing for each monoclonal antibody-HIV-1 Env binding interactions is used to subtract nonspecific binding and signal drift of the Env proteins to the control surface and blank buffer flow, respectively. Antibody capture level on the sensor surface is optimized for each monoclonal antibody to minimize rebinding and any associated avidity effects. C.CH505 Env gpl40 protein is injected at concentrations ranging from 2 to 25 μg ml-1, and B.63521 gpl20 was injected at 50-400 μg ml-1 for UCAs and early intermediates IA8 and IA4, 10-100 μg ml-1 for intermediate IA3, and 1- 25 μg ml-1 for the distal and mature monoclonal antibodies. All curve-fitting analyses are performed using global fit of to the 1 : 1 Langmuir model and are representative of at least three measurements. All data analysis was performed using the BIAevaluation 4.1 analysis software (GE Healthcare).

[0086] Neutralization assays [0087] Neutralizing antibody assays in TZM-bl cells are performed as described previously.

Neutralizing activity of plasma samples in eight serial threefold dilutions starting at 1 :20 dilution and for recombinant monoclonal antibodies in eight serial threefold dilutions starting at 50 μg ml-1 are tested against autologous and herologous HIV-1 Env-pseudotyped viruses in TZM-bl-based neutralization assays using the methods known in the art. Neutralization breadth of CHI 03 is determined using a panel of 196 of geographically and genetically diverse Env-pseudoviruses representing the major circulated genetic subtypes and circulating recombinant forms. HIV-1 subtype robustness is derived from the analysis of HIV-1 clades over time. The data are calculated as a reduction in luminescence units compared with control wells, and reported as IC50 in either reciprocal dilution for plasma samples or in micrograms per microlitre for monoclonal antibodies.

[0088] The GenBank accession numbers for 292 CH505 Env proteins are KC247375-KC247667, and accessions for 459 VHDJ _h and 174 VLJL sequences of antibody members in the CHI 03 clonal lineage are KC575845-KC576303 and KC576304-KC576477, respectively..

Example 2

[0089] Binding of sequential envelopes to CHI 03 and CH235 CD4 binding site bnAb lineages members.

[0090] The binding assay was an ELISA with the envelope protein bound to the well surface of a 96 well plate, and the antibody in questions incubated with the envelope bound to the plate. After washing, an enzyme- labeled anti-human IgG antibody was added and after incubation, washed away. The intensity of binding was determined by the intensity of enzyme-activated color in the well.

[0091] Table 1. ELISA binding, log-transformed area under the curve (AUC) values for a realization with four Env-derived gpl20 antigens, assayed against members of the CH103 bnAb lineage from universal ancestor (UCA), through intermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0 indicate no binding. The transmitted-founder (TF) antigen was derived from Env w004.3.

Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2 CH104 IA1 CH106 CH103

TF 3.5 5.5 9.2 9.1 10.1 11 11.2 10.8 10.4 10.4 11.3 12.6 w053.16 0 0 0 0 0.2 1.1 9 9.3 9.9 8.8 9.8 11.6 w078.33 0 0 0 0 0 0 8.9 9 9 8.2 9.5 11.1 wl00.B6 0 0 0 0 0 0 11 12.1 11 12.2 11.8 7.1 [0092] Table 2. ELISA binding, log-transformed area under the curve (AUC) values for a realization with five Env-derived gpl20 antigens, assayed against members of the CH103 bnAb lineage from universal ancestor (UCA), through intermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0 indicate no binding. Antigen names beginning with M were synthesized by site-directed mutagenesis.

Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2 CH104 IA1 CH106 CH103

Mi l 2.6 6.2 10.1 10 10.5 11.8 11.7 12.7 12 12.2 12.8 13.4

M5 0 0.6 2.3 3.3 3.8 6.8 8.6 7.8 9 7 8.4 9.8 w020.14 0.3 3.4 7.2 7.9 8.6 9.5 10.4 11 10.4 10.3 11.2 12.6 w030.28 0 1.6 3.5 6.3 6.5 7.7 9.1 11.1 10.7 10.1 11.7 12.8 w078.15 0 0 0.7 1 1.3 3 10.1 11.5 10.8 10.9 11 10.7 w053.31 0 0 0 0 0 0 13.5 13.3 13.7 13.4 13.4 13.6

[0093] Table 3. ELISA binding, log-transformed area under the curve (AUC) values for a realization with five Env-derived gpl20 antigens, assayed against members of the DH235 (CH235) bnAb helper lineage from universal ancestor (UCA), through intermediate ancestors (14-11) to mature bnAbs. Values of 0 indicate no binding. Antigen names beginning with M were synthesized by site-directed mutagenesis.

Antigen UCA 14 13 12 11 DH235 CH236 CH239 CH240 CH241

Mi l 0 0 0 0 2.8 7.6 1.4 1.4 0.5 9.7

M5 0.2 1.4 7 6.9 9.2 11.4 7.3 12.9 7.4 14.5 w020.14 0 0 2.7 1.2 6.5 9.9 6.7 9 3.8 13.1 w030.28 0 0 0 0 2.4 6.7 1.5 3.6 0.3 9.6 w078.15 0 0 0 0 0 0 0 0 0 0 w053.31 0 0 0 0 0 1.1 0 0 0 1.4

[0094] Table 4. ELISA binding, log-transformed area under the curve (AUC) values for a realization that embodies ten Env-derived gpl20 antigens, assayed against members of the CH103 bnAb lineage from universal ancestor (UCA), through intermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0 indicate no binding. Antigen names beginning with M were synthesized by site-directed mutagenesis.

Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2 CH104 IA1 CH106 CH103

Mi l 2.6 6.2 10.1 10 10.5 11.8 11.7 12.7 12 12.2 12.8 13.4 M5 0 0.6 2.3 3.3 3.8 6.8 8.6 7.8 9 7 8.4 9.8 w020.14 0.3 3.4 7.2 7.9 8.6 9.5 10.4 11 10.4 10.3 11.2 12.6 w030.28 0 1.6 3.5 6.3 6.5 7.7 9.1 11.1 10.7 10.1 11.7 12.8 w078.15 0 0 0.7 1 1.3 3 10.1 11.5 10.8 10.9 11 10.7 w053.16 0 0 0 0 0.2 1.1 9 9.3 9.9 8.8 9.8 11.6 w030.21 0 0 0 0 0 0 10.6 11.5 11.3 11.8 10.9 12.2 w078.33 0 0 0 0 0 0 8.9 9 9 8.2 9.5 11.1 wl00.B6 0 0 0 0 0 0 11 12.1 11 12.2 11.8 7.1 w053.31 0 0 0 0 0 0 13.5 13.3 13.7 13.4 13.4 13.6

Example 3

[0095] Combinations of antigens derived from CH505 envelope sequences for swarm

immunizations

[0096] Provided herein are non-limiting examples of combinations of antigens derived from CH505 envelope sequences for a swarm immunization. Without limitations, these selected combinations comprise envelopes which provide representation of the sequence and antigenic diversity of the HIV-1 envelope variants which lead to the induction and maturation of the CHI 03 and CH235 antibody lineages. The identification of bnAb lineage (CHI 03) and envelopes which bind preferentially to various members of this lineage provides a direct strategy for the selection of Envs (out of millions possible envelopes naturally occurring in an HIV-1 infected individual) that might have engaged UCA and participated in bnAb development, and thus could serve as immunogens in a vaccine formulation. The identification of helper lineage (CH235) and envelopes which bind preferentially to various members this lineage provides a direct strategy for the selection of Envs (out of millions possible envelopes naturally occurring in an HIV-1 infected individual) that might have engaged UCA and participated in bnAb development, and thus could serve as immunogens in a vaccine formulation.

[0097] The selection includes priming with a virus which binds to the UCA, for example a T/F virus or another early (e.g. but not limited to week 004.3, or 004.26) virus envelope. In certain embodiments the prime could include D-loop variants. In certain embodiments the boost could include D-loop variants. In certain embodiments, these D-loop variants are envelope escape mutants not recognized by the UCA. Non-limiting examples of such D-loop variants are envelopes designated as M10, Ml 1, M19, M20, M21, M5, M6, M7, M8, M9, M14 (TF_M14), M24 (TF_24), M15, M16, M17, M18, M22, M23, M24, M25, M26. See Gao et al. (2014) Cell 158:481-491. [0098] Non-limiting embodiments of envelopes selected for swarm vaccination are shown as the selections described below. A skilled artisan would appreciate that a vaccination protocol can include a sequential immunization starting with the "prime" envelope(s) and followed by sequential boosts, which include individual envelopes or combination of envelopes. In another vaccination protocol, the sequential immunization starts with the "prime" envelope(s) and is followed with boosts of cumulative prime and/or boost envelopes. In certain embodiments, the sequential immunization starts with the "prime" envelope(s) and is followed by boost(s) with all or various combinations of the envelopes in the selection. In certain embodiments, the prime does not include T/F sequence (W000.TF). In certain embodiments, the prime includes w004.03 envelope. In certain embodiments, the prime includes w004.26 envelope. In certain embodiment the prime includes Ml 1. In certain embodiments the prime includes M5. In certain embodiments, the immunization methods do not include immunization with HIV-1 envelope T/F. In certain embodiments, the immunization methods do not include a schedule of four valent immunization with HIV-1 envelopes T/F, w053.16, w078.33, and wl00.B6.

[0099] In certain embodiments, there is some variance in the immunization regimen; in some embodiments, the selection of HIV-1 envelopes may be grouped in various combinations of primes and boosts, either as nucleic acids, proteins, or combinations thereof.

[0100] In certain embodiments the immunization includes a prime administered as DNA, and MVA boosts. See Goepfert, et al. 2014; "Specificity and 6-Month Durability of Immune Responses Induced by DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus- Like Particles" J Infect Dis. 2014 Feb 9. [Epub ahead of print].

[0101] HIV-1 Envelope selection A (five envelopes): Mi l; w020.14; w030.28; w078.15;

W053.31

[0102] HIV-1 Envelope selection B (six envelopes): Mi l; M5; w020.14; w030.28; w078.15; W053.31

[0103] HIV-1 Envelope selection C (ten envelopes): Mi l; M5; w020.14; w030.28; w078.15; W053.16; w030.21; w078.33; wl00.B6; w053.31.

[0104] The selections of CH505-Envs were down-selected from a series of 400 CH505 Envs isolated by single-genome amplification followed for 3 years after acute infection, based on experimental data. The enhanced neutralization breadth that developed in the CD4-binding site (bs) CHI 03 antibody lineage that arose in subject CH505 developed in conjunction with epitope diversification in the CH505's viral quasispecies. It was observed that at 6 months post-infection in there was more diversification in the CD4bs epitope region in this donor than sixteen other acutely infected donors. Population breadth did not arise in the CHI 03 antibody lineage until the epitope began to diversify. A hypothesis is that the CHI 03 linage drove viral escape, but then the antibody adapted to the relatively resistant viral variants. As this series of events was repeated, the emerging antibodies evolved to tolerate greater levels of diversity in relevant sites, and began to be able to recognize and neutralize diverse heterologous forms for the virus and manifest population breadth. In certain embodiments, six envs are selected from CH505 sequences to reflect diverse variants for making Env pseudoviruses, with the goal of recapitulating CH505 HIV-1 antigenic diversity over time, making sure selected site (i.e. those sites reflecting major antigenic shifts) diversity was represented.

[0105] Specifically, for CH505 the virus and envelope evolution were mapped, and the CHI 03 CD4 binding-site bnAb evolution. In addition, 135 CH505 varied envelope pseudotyped viruses were made and tested them for neutralization sensitivity by members of the CHI 03 bnAb lineage (e.g, Figures 3). From this large dataset, in one embodiment, six Env variants were chosen for immunization based on sequence diversity, and antigenic diversity, for example binding to antibodies in the CHI 03 and/or CH235 lineage (Tables 2-4).

[0106] In certain embodiments, the envelopes are selected based on Env mutants with sites under diversifying selection, in which the transmitted/founder (T/F) Env form vanished below 20% in any sample, i.e. escape variants; signature sites based on autologous neutralization data, i.e. Envs with statistically supported signatures for escape from members of the CHI 03 bnAb lineage; and sites with mutations at the contact sites of the CHI 03 antibody and HIV Env. In this manner, a sequential swarm of Envs was selected for immunization to represent the progression of virus escape mutants that evolved during bnAb induction and increasing neutralization breadth in the CH505 donor.

[0107] In certain embodiments, additional sequences are selected to contain five additional specific amino acid signatures of resistance that were identified at the global population level. These sequences contain statistically defined resistance signatures, which are common at the population level and enriched among heterologous viruses that CH103 fails to neutralize. When they were introduced into the TF sequence, they were experimentally shown to confer partial resistance to antibodies in the CHI 03 lineage. Following the reasoning that serial viral escape and antibody adaptation to escape is what ultimate selects for neutralizing antibodies that exhibit breadth and potency against diverse variants, in certain embodiments, inclusion of these variants in a vaccine may extend the breadth of vaccine-elicited antibodies even beyond that of the CHI 03 lineage. Thus the overarching goal will be to trigger a CH103-like lineage first using the CH505TF modified Ml 1, that is well recognized by early CHI 03 ancestral states, then vaccinating with antigenic variants, to allow the antibody lineage to adapt through somatic mutation to accommodate the natural variants that arose in CH505. In certain embodiments, vaccination regimens include a total of five sequences (Selection A) that capture the antigenic diversity of CH505. In another embodiment, additional antigenic diversity is added (Selection B and C), to enable the induction of antibodies by vaccination that may have even greater breadth than those antibodies isolated from CH505.

[0108] In some embodiments, the CH505 sequences that represent the accumulation of viral sequence and antigenic diversity in the CD4bs epitope of CH103 in subject CH505 are represented by selection A, selection B, or selection C.

[0109] Ml 1 is a mutant generated to include two mutations in the loop D (N279D + V281G relative to the TF sequence) that enhanced binding to the CHI 03 lineage (see Figure 29). These were early escape mutations for another CD4bs autologous neutralizing antibody lineage, but might have served to promote early expansion of the CHI 03 lineage.

[0110] In certain embodiments, the two CHI 03 resistance signature-mutation sequences added to the antigenic swarm are: M14 (TF with S364P), and M24 (TF with S375H + T202K + L520F + G459E). They confer partial resistance to the TF with respect to the CHI 03 lineage. In certain embodiments, these D-loop mutants are administered in the boost.

Example 4

[0111] Immunization protocols in subjects with swarms of HIV-1 envelopes.

[0112] Immunization protocols contemplated by the invention include envelopes sequences as described herein including but not limited to nucleic acids and/or amino acid sequences of gpl60s, gpl50s, gpl45, cleaved and uncleaved gpl40s, gpl20s, gp41s, N-terminal deletion variants as described herein, cleavage resistant variants as described herein, or codon optimized sequences thereof. A skilled artisan can readily modify the gpl60 and gpl20 sequences described herein to obtain these envelope variants. The swarm immunization selections can be administered in any subject, for example monkeys, mice, guinea pigs, or human subjects.

[0113] In non-limiting embodiments, the immunization includes a nucleic acid which is administered as DNA, for example in a modified vaccinia vector (MVA). In non-limiting embodiments, the nucleic acids encode gpl60 envelopes. In other embodiments, the nucleic acids encode gpl20 envelopes. In other embodiments, the boost comprises a recombinant gpl20 envelope. The vaccination protocols include envelopes formulated in a suitable carrier and/or adjuvant, for example but not limited to alum. In certain embodiments the immunizations include a prime, as a nucleic acid or a recombinant protein, followed by a boost, as a nucleic acid or a recombinant protein. A skilled artisan can readily determine the number of boosts and intervals between boosts.

[0114] Table I shows a non-limiting example of an immunization protocol using a selection of HIV-1 envelopes

[0115] Table II shows a non-limiting example of an immunization protocol using a selection of HIV-1 envelopes

vector and/or vector and/or vector and/or vector and/or protein protein protein protein

W020.14 W020.14 W020.14 W020.14

as a nucleic acid as a nucleic acid as a nucleic acid e.g. DNA/MVA e.g. DNA/MVA e.g. DNA/MVA and/or protein and/or protein and/or protein

W030.28 W030.28 W030.28

as nucleic acid as nucleic acid e.g. DNA/MVA e.g. DNA/MVA and/or protein and/or protein

W078.15 W078.15 W078.15

as nucleic acid as nucleic acid e.g. DNA/MVA e.g. DNA/MVA and/or protein and/or protein

W100.B6 W100.B6

as nucleic acid e.g. DNA/MVA and/or protein

[0116] In certain embodiments, after administering a prime with Ml 1, subsequent immunizations include all other envelopes as nucleic acids and/or proteins.

[0117] Table III shows a non-limiting example of an immunization protocol using a swarm of

HIV-1 envelopes

and/or

protein

W020.14 W020.14

as a nucleic

acid e.g.

DNA/MVA

and/or protein

W030.28 W030.28

as nucleic acid

e.g.

DNA/MVA

and/or protein

W078.15 W078.15

as nucleic acid

e g-

DNA/MVA

and/or protein

W100.B6 W100.B6 as nucleic acid e g-

DNA/MVA and/or protein 18] Table IV shows a non-limiting example of an immunization protocol using a swarm of HIV-1 envelopes

M5 Optionally M5 M5 M5 M5

M5 as a nucleic as a nucleic as a nucleic as a nucleic as a nucleic acid e.g. acid e.g. acid e.g. acid e.g. acid e.g. DNA/MVA DNA/MVA DNA/MVA DNA/MVA DNA/MVA and/or and/or protein and/or protein and/or protein and/or protein protein

W020.14 W020.14 W020.14 W020.14 as a nucleic as a nucleic as a nucleic acid e.g. acid e.g. acid e.g. DNA/MVA DNA/MVA DNA/MVA and/or protein and/or protein and/or protein

W030.28 W030.28 W030.28 as nucleic as nucleic acid e.g. acid e.g. DNA/MVA DNA/MVA and/or protein and/or protein

W078.15 W078.15 W078.15 as nucleic as nucleic acid e.g. acid e.g. DNA/MVA DNA/MVA and/or protein and/or protein

W100.B6 W100.B6 as nucleic acid e.g. DNA/MVA and/or protein

[0119] In certain embodiments, after administering a prime with Ml 1 and optionally with M5, subsequent immunizations include all other envelopes as nucleic acids and/or proteins.

[0120] Table V shows a non-limiting example of immunization protocol using a selection of ten HIV-1 envelopes Mi l Mi l as a

nucleic

acid e.g.

DNA/M

VA

vector

and/or

protein

M5 M5

as a

nucleic

acid e.g.

DNA/M

VA

and/or

protein

W020. W020.14

14 as a

nucleic acid e.g.

DNA/M

VA

and/or

protein

W030. W030.28 28 as nucleic acid e.g.

DNA/M

VA

and/or protein

W078. W078.15 15 as nucleic acid e.g.

DNA/M

VA

and/or protein W053. W053.16

16 as

nucleic

acid e.g.

DNA M

VA

and/or

protein

W030. W030.21

21 as

nucleic

acid e.g.

DNA M

VA

and/or

protein

W078. W078.33

33 as

nucleic

acid e.g.

DNA/M

VA

and/or

protein

W100. W100.B

B6 6

as

nucleic acid e.g.

DNA/M

VA

and/or

protein

W053. W053.31 31 as nucleic acid e.g. DNA/M VA

and/or protein

[0121] In certain embodiments, after administering a prime with Ml 1 and optionally with M5, subsequent immunizations include cumulative addition of the other envelopes as nucleic acids and/or proteins.

[0122] In certain embodiments, after administering a prime with Ml 1 and optionally with M5, subsequent immunizations include all other envelopes as nucleic acids and/or proteins.

[0123] In certain embodiments an immunization protocol could prime with a bivalent or trivalent Gag mosaic (Gagl and Gag 2, Gag 1, Gag 2 and Gag3) in a suitable vector.

Example 5

[0124] Env mixtures of the CH505 virus are expected to induce the beginning of CD4 binding site BnAb lineages CH103 and CH235

[0125] The combinations of envelopes described in Examples 2-4 will be tested in any suitable subject. Suitable animal models include without limitation mice, including humanized mice, guinea pigs, or non-human primates ( HPs) . For example an animal is administered with the following antigens in the following immunization schedule: Prime: loop D mutant M5 and Ml 1. That will give us the best CH103 UCA binder (Ml 1) and the best CH235 UCA binder (M5). Immunization 2: week 020.14. Immunization 3 : Week 030.28. Immunization 4: week 078.15. Immunization 5: week 100.B6. Immunization 5: swarm of all six envelopes. Adjuvant is a TLR-4 agonist (GLA- synthetic monophosphoryl lipid A) in stable emulsion from Infectious Disease Research Institute, Seattle WA.

Example 6:

[0126] One of the major obstacles to developing an efficacious preventive HIV-1 vaccine is the challenge of inducing broadly neutralizing antibodies (bnAbs) against the virus. There are several reasons why eliciting bnAbs has been challenging and these include the conformational structure of the viral envelope, molecular mimicry of host antigens by conserved epitopes which may lead to the suppression of potentially useful antibody responses, and the high level of somatic mutations in the variable domains and the requirement for complex maturation pathways [1-3]. It has been shown that up to 25% of HIV-1- infected individuals develop bnAbs that are detected 2-4 years after infection. To date, all bnAbs have one or more of these unusual antibody traits: high levels of somatic mutation, autoreactivity with host antigens, and long heavy chain third complementarity determining regions (HCDR3s)— all traits that are controlled or modified by host

immunoregulatory mechanisms. Thus, the hypothesis has been put forth that typical vaccinations of single primes and boosts will not suffice to be able to induce bnAbs; rather, it will take sequential immunizations with Env immunogens, perhaps over a prolonged period of time, to mimic bnAb induction in chronically infected individuals [4].

[0127] A process to circumvent host immunoregulatory mechanisms involved in control of bnAbs is termed B cell lineage immunogen design, wherein sequential Env immunogens are chosen that have high affinities for the B cell receptors of the unmutated common ancestor (UCA) or germline gene of the bnAb clonal lineage [4]. Envs for immunization can either be picked randomly for binding or selected, as described herein, from the evolutionary pathways of Envs that actually give rise to bnAbs in vivo. Liao and colleagues recently described the co-evolution of HIV-1 and a CD4 binding site bnAb from the time of seroconversion to the development of plasma bnAb induction, thereby presenting an opportunity to map out the pathways that lead to generation of this type of CD4 binding site bnAb [5]. They showed that the single transmitted/founder virus was able to bind to the bnAb UCA, and identified a series of evolved envelope proteins of the founder virus that were likely stimulators of the bnAb lineage. Thus, this work presents an opportunity to vaccinate with naturally-derived viral envelopes that could drive the desired B-cell responses and induce the development of broad and potent neutralizing antibodies. While the human antibody repertoire is diverse, it has been found that only a few types of B cell lineages can lead to bnAb development, and that these lineages are similar across a number of individuals [6,7]. Thus, it is feasible that use of Envs from one individual will generalize to others.

[0128] In certain embodiments the invention provides methods for selecting the Env immunogens, among multitude of diverse viruses that induced a CD4 binding site bnAb clonal lineage in an HIV- infected individual, by making sequential recombinant Envs from that individual and using these Envs for vaccination. The B-cell lineage vaccine strategy thus includes designing immunogens based on unmutated ancestors as well as intermediate ancestors of known bnAb lineages. A candidate vaccine could use transmitted/founder virus envelopes to, at first, stimulate the beginning stages of a bnAb lineage, and subsequently boost with evolved Env variants to recapitulate the high level of somatic mutation needed for affinity maturation and bnAb activity. The goal of such a strategy is to selectively drive desired bnAb pathways.

[0129] Broadly neutralizing antibodies likely will not be induced by a single Env, and even a mixture of polyvalent random Envs (e.g. HVTN 505) is unlikely to induce bnAbs. Rather, immunogens must be designed to trigger the UCAs of bnAb lineages to undergo initial bnAb lineage maturation, and then use sequential immunogens to fully expand the desired lineages. The proposed trial will represent the first of many experimental clinical trials testing this concept in order to develop the optimal set of immunogens to drive multiple specificities of bnAbs. The HVTN will be at the cutting edge of this effort.

[0130] The concept is applicable to driving CD4 binding site lineage in multiple individuals due to the convergence of a few bnAb motifs among individuals. The adjuvant will be the GSK AS01E adjuvant containing MPL and QS21. Other suitable adjuvants can be used. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic using HBsAg as vaccine antigen [Leroux-Roels et al., IABS Conference, April 2013, [9].

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