Recombinant HIV-1 envelope proteins and their use
Inventors
Kwong, Peter • Pancera, Marie • Zhou, Tongqing • Georgiev, Ivelin • Joyce, Michael Gordon • Acharya, Priyamvada • Gorman, Jason • Yang, YongPing • Druz, Aliaksandr • Stewart-Jones, Guillaume • Chen, Rita • Chuang, Gwo-Yu • Baxa, Ulrich • Mascola, John • Lynch, Rebecca • Zhang, Baoshan • Cheng, Cheng
Assignees
US Department of Health and Human Services
Publication Number
US-10400015-B2
Publication Date
2019-09-03
Expiration Date
2035-09-04
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Abstract
HIV-1 Env ectodomain trimers stabilized in a prefusion mature closed conformation and methods of their use and production are disclosed. In several embodiments, the HIV-1 Env ectodomain trimers and/or nucleic acid molecules can be used to generate an immune response to HIV-1 in a subject. In additional embodiments, the therapeutically effective amount of the HIV-1 Env ectodomain trimers can be administered to a subject in a method of treating or preventing HIV-1 infection.
Core Innovation
The invention provides recombinant HIV-1 Env ectodomain trimers stabilized in a prefusion mature closed conformation, a conformation that evades antibody recognition and is the precursor to membrane fusion. The atomic-level three-dimensional structure of the HIV-1 Env ectodomain in this conformation is disclosed, showing that it comprises three gp120-gp41 protomers with a V1V2 domain cap and distinct α6 and α7 helices at the membrane proximal aspect. The structure reveals how gp41 forms a 4-helix collar enclosing the N- and C-termini strands of gp120, stabilized by non-natural disulfide bonds and amino acid substitutions, which prevent conformational rearrangements that lead to the CD4-bound open state.
The problem addressed is the immunoevasion by the HIV-1 Env ectodomain trimer, which, despite eliciting Env-reactive antibodies, successfully evades most antibody-mediated neutralization largely owing to structural rearrangements from the prefusion mature closed conformation to the CD4-bound open conformation. The absence of atomic-level structural information of this closed conformation has hindered the design of immunogens capable of stably presenting this vulnerable and broadly neutralizing epitope. The invention solves this by using the disclosed atomic structure to design recombinant HIV-1 Env ectodomain trimers and chimeric variants that resist transition to the open conformation even in the presence of CD4, thus maintaining exposure of epitopes targeted by broadly neutralizing antibodies.
The disclosed recombinant HIV-1 Env ectodomain trimers can include modifications such as non-natural disulfide bonds between cysteine substitutions at defined HIV-1 Env positions (e.g., positions 201 and 433, 501 and 605), proline substitutions (e.g., at position 559), cavity-filling amino acid substitutions, and introduced glycosylation sites. These trimers can specifically bind broadly neutralizing antibodies like PGT122, VRC26, and PGT145, but do not bind poorly neutralizing antibodies such as 17b in the presence of soluble CD4, indicating stabilization in the prefusion mature closed conformation. Chimeric trimers incorporating sequences from multiple HIV-1 strains (including V1V2 domains from diverse strains transplanted onto BG505 platforms) are designed to address HIV-1 sequence diversity. Methods of use include generating immune responses in subjects by administering these stabilized trimers or fragments thereof, as protein nanoparticles or encoded by nucleic acid molecules or viral vectors.
Claims Coverage
The patent includes independent claims directed to isolated immunogens comprising recombinant HIV-1 Env ectodomain trimers stabilized in a prefusion mature closed conformation with specific amino acid substitutions, particularly non-natural disulfide bonds, and related nucleic acids, vectors, host cells, compositions, and methods of use.
Recombinant HIV-1 Env ectodomain trimer stabilized in prefusion mature closed conformation
An isolated immunogen comprising a recombinant HIV-1 Env ectodomain trimer stabilized in a prefusion mature closed conformation by one or more amino acid substitutions compared to native HIV-1 Env sequence, including cysteine substitutions at positions 201 and 433 to form a non-natural disulfide bond, resistant to binding by 17b antibody in the presence of excess sCD4; comprising three gp120-gp41 protomers.
Definition of recombinant Env protein boundaries
The recombinant HIV-1 Env ectodomain trimer includes a gp120 polypeptide and a gp41 ectodomain with defined N- and C-terminal residue boundaries corresponding to HXB2 positions 1-35 to 503-511 for gp120, and 512-522 to 624-705 for gp41 ectodomain.
Chimeric Env ectodomain trimers with sequences from multiple HIV-1 strains
Trimers comprising gp41 ectodomain and terminal regions of gp120 from a first strain of HIV-1, with the remaining gp120 sequence from a heterologous strain; sequences modified to include stabilizing amino acid substitutions preserving the prefusion mature closed conformation.
Specific stabilizing amino acid substitutions for conformation fixation
Inclusion of specific amino acid substitutions such as cavity-filling substitutions, non-natural disulfide bonds between cysteine substitutions at positions 501 and 605, proline substitution at position 559, and glycosylation site introduction at specific positions to stabilize the mature closed conformation.
Single chain HIV-1 Env ectodomains
Trimers comprising gp120-gp41 protomers as single chain HIV-1 Env ectodomains, where gp120 and gp41 are covalently linked by peptide linkers at defined positions; may include stabilizing amino acid substitutions as described.
Linkage to membrane anchor and trimerization domains
Recombinant HIV-1 Env ectodomain trimers linked to transmembrane domains or trimerization domains such as Foldon domains to provide membrane anchoring or promote trimerization, without disrupting prefusion mature closed conformation.
Nucleic acid molecules, vectors, host cells and compositions
Nucleic acid molecules encoding the stabilized trimers, operably linked to promoters, vectors comprising these nucleic acids, host cells transformed therewith, and immunogenic compositions including the trimers or nucleic acids, optionally with adjuvants, for inducing immune responses.
Therapeutic and diagnostic methods
Methods of generating an immune response to HIV-1 by administering the recombinant stabilized Env trimers or corresponding compositions, diagnosing or isolating HIV-1 Env-specific antibodies by contacting biological samples with the immunogen, and methods of treating or preventing HIV-1 infection in subjects at risk or infected.
The patent claims cover isolated immunogens comprising recombinant HIV-1 Env ectodomain trimers stabilized in the prefusion mature closed conformation using specific amino acid substitutions including non-natural disulfide bonds. The claims also cover chimeric versions incorporating diverse HIV-1 strain sequences, single-chain formats, linkage to membrane anchors and trimerization domains, nucleic acid molecules and vectors encoding these immunogens, compositions with adjuvants, and methods of use for generating immune responses, therapy, diagnosis, and antibody detection.
Stated Advantages
The recombinant HIV-1 Env ectodomain trimers resist transition to the CD4-bound open conformation, retaining the prefusion mature closed conformation and thus preventing exposure of poorly neutralizing antibody epitopes and maximizing exposure of broadly neutralizing antibody epitopes.
Stabilized trimers show improved physical stability to extremes of temperature, pH, osmolarity, and freeze-thaw cycles, which is desirable for manufacturing and storage.
The immunogens retain specific binding to broadly neutralizing antibodies targeting the prefusion closed conformation and exhibit reduced binding to ineffective antibodies, especially under CD4 presence, enhancing antigenic specificity.
Chimeric immunogens incorporating diverse HIV-1 strain sequences can address HIV-1 sequence diversity, improving breadth of induced immune responses.
Single-CD4 binding state observed with stabilized trimers reveals new mechanistic insights into HIV-1 entry conformations, supporting vaccine design.
Documented Applications
Use of recombinant HIV-1 Env ectodomain trimers stabilized in the prefusion mature closed conformation as immunogens to generate an immune response and to induce neutralizing antibodies to HIV-1 in a subject.
Methods of treating or preventing HIV-1 infection in a subject by administering therapeutically effective amounts of the stabilized recombinant trimers, protein nanoparticles including such trimers, nucleic acid molecules encoding such trimers, or viral vectors including such nucleic acids.
Use in prime-boost vaccination protocols employing nucleic acid vectors encoding the trimers as primes and protein or nanoparticle trimers as boosts.
Diagnostic methods for detecting or isolating HIV-1 Env-specific antibodies in samples by contacting with the stabilized recombinant HIV-1 Env ectodomain trimer immunogen.
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