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 • Stewart-Jones, Guillaume • Chen, Rita • Chuang, Gwo-Yu • Mascola, John • Zhang, Baoshan • Cheng, Cheng • Sastry, Mallika • Druz, Aliaksandr
Assignees
US Department of Health and Human Services
Publication Number
US-11459360-B2
Publication Date
2022-10-04
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
HIV-1 Env ectodomain trimers are key components facilitating virus entry into host cells by interacting with host cellular receptors and fusing viral and host-cell membranes. However, despite their exposed position, these trimers evade antibody-mediated neutralization, complicating HIV vaccine development. The Env trimer comprises three gp120-gp41 protomers with extensive glycosylation and undergoes conformational transitions from a prefusion mature closed conformation to CD4- and co-receptor-bound conformations, culminating in a postfusion state.
A primary challenge has been stabilizing recombinant HIV-1 Env ectodomain trimers in the prefusion mature closed conformation, recognized by broadly neutralizing antibodies, to serve as effective immunogens. The lack of detailed atomic structures of the Env trimer in this conformation hindered structure-guided design efforts to create stabilized immunogens that maintain this closed state even in the presence of CD4.
This disclosure provides, for the first time, high-resolution atomic-level three-dimensional structures of the HIV-1 Env ectodomain trimer in the prefusion mature closed conformation, alone and complexed with broadly neutralizing antibodies PGT122 and 35O22. These structures reveal structural features including distinct helices in gp41, quaternary interactions, and glycan shielding that contribute to the mature quaternary structure and immune evasion. The understanding of conformational rearrangements and immune recognition vulnerabilities enabled the design of recombinant Env ectodomain trimers stabilized by specific amino acid substitutions that resist transition to the CD4-bound open conformation and retain the prefusion mature closed conformation when incubated with CD4.
Claims Coverage
The patent claims encompass multiple inventive features related to recombinant HIV-1 Env ectodomain trimers stabilized in a prefusion mature closed conformation.
Recombinant HIV-1 Env ectodomain trimer stabilized in prefusion mature closed conformation
An isolated immunogen comprising a recombinant HIV-1 Env ectodomain trimer with three gp120-gp41 protomers, having one or more amino acid substitutions that stabilize the trimer in the prefusion mature closed conformation, specifically having an α7 helix formed after position 570, a distance of less than five angstroms between positions 200 and 313 on gp120, and that does not specifically bind to a CD4-induced antibody in the presence of molar excess sCD4.
Defined boundaries for gp120 and gp41 in recombinant Env ectodomain
The Env ectodomain trimer comprises a gp120 polypeptide with an N-terminal residue between positions 1-35 and a C-terminal residue between positions 503-512; a gp41 ectodomain with an N-terminal residue between 512-522 and a C-terminal residue between 624-705, corresponding to the HXB2 reference sequence.
Cysteine substitutions to form non-natural disulfide bond stabilizing Env trimer
The recombinant HIV-1 Env ectodomain trimer includes cysteine substitutions forming non-natural disulfide bonds that stabilize the trimer in the prefusion mature closed conformation; including disulfide bonds between cysteines substituted at positions within V2 domain and β21 sheet, or positions 195-201 and 423-433, or specifically at positions 120 and 315, 195 and 433, 199 and 433, or 425 and 433 (HXB2 numbering).
Proline substitutions inhibiting α0 helix formation for stabilization
Inclusion of a proline substitution at position 66 and/or 67 of the gp120 polypeptide inhibits formation of the α0 helix, contributing to stabilization of the Env ectodomain trimer prefusion mature closed conformation.
Cavity filling amino acid substitutions for stabilization
The recombinant trimer includes cavity filling amino acid substitutions at specified HXB2 positions to reduce internal cavities that collapse upon conformational transition, thereby stabilizing the prefusion mature closed conformation.
Combinations including gp41 stabilizing mutations and SOSIP
The one or more amino acid substitutions may include mutations that stabilize gp41 (e.g., cysteine substitutions to introduce disulfide bonds between positions 501 and 605 and a proline substitution at position 559), optionally combined with cavity filling or other mutations and SOS/N mutations.
Chimeric Env ectodomains from multiple HIV-1 strains
The recombinant Env ectodomain trimers may be chimeric, including sequences from at least two or three HIV-1 strains, with the gp41 ectodomain and N- and C-terminal regions of gp120 from one strain; the V1V2 domain from a second strain; and the remainder of gp120 from another strain, all including stabilizing substitutions.
Single chain Env ectodomain with peptide linker
The gp120 and gp41 protomers can be linked as a single chain Env ectodomain with the N-terminal residue of gp120 linked to the C-terminal residue of gp41 by a heterologous peptide linker.
Linkage to trimerization or transmembrane domains
The Env ectodomain trimer can be linked to a trimerization domain, such as a Foldon domain, and/or to a transmembrane domain to enable membrane anchoring.
Protein nanoparticles including stabilized HIV-1 Env ectodomain trimers
Self-assembling protein nanoparticles can include the recombinant HIV-1 Env ectodomain trimers stabilized in the prefusion mature closed conformation.
The claims cover recombinant HIV-1 Env ectodomain trimers stabilized in the prefusion mature closed conformation by amino acid substitutions including non-natural disulfide bonds, cavity filling, and proline substitutions. The trimer structure is defined with specified boundaries and conformational features, and may be chimeric across multiple strains. Further claims encompass single-chain forms, linkage to trimerization or transmembrane domains, inclusion in protein nanoparticles, nucleic acid molecules encoding such trimers, vectors, and methods of generating immune responses and treatment.
Stated Advantages
Stabilization of the HIV-1 Env ectodomain trimers in the prefusion mature closed conformation resists transition to the CD4-bound open conformation when incubated with CD4, avoiding exposure of antigenic sites targeted by poorly neutralizing antibodies and maximizing exposure of sites targeted by broadly neutralizing antibodies.
The recombinant HIV-1 Env ectodomain trimers show improved antigenic specificity, including exclusive exposure of neutralizing epitopes, even in the presence of CD4.
The DS substitution (201C-433C) increases thermostability and temporal stability of the Env trimer, making it a highly desirable immunogen.
The single CD4-bound state observed for the stabilized trimers reveals an obligatory intermediate in HIV-1 entry and provides mechanistic insights.
Recombinant trimers stabilized in the prefusion mature closed conformation induce neutralizing immune responses in animal models.
Documented Applications
Use of stabilized recombinant HIV-1 Env ectodomain trimers and related compositions to generate an immune response to HIV-1 in a subject.
Methods of treating, inhibiting, or preventing HIV-1 infection in a subject by administering a therapeutically effective amount of the stabilized recombinant HIV-1 Env ectodomain trimers or immunogenic fragments thereof.
Use of recombinant HIV-1 Env ectodomain trimers as immunogens in vaccine compositions, including prime-boost vaccination regimens.
Use of recombinant HIV-1 Env ectodomain trimer-containing protein nanoparticles or virus-like particles for immunization to elicit neutralizing antibodies.
Use of recombinant HIV-1 Env ectodomain trimers and constructs in diagnostic assays to detect antibodies specific to HIV-1.
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