Recombinant HIV-1 envelope proteins and their use
Inventors
Kwong, Peter • Mascola, John • Chuang, Gwo-Yu • Geng, Hui • Yang, YongPing • Cheng, Cheng • Boyington, Jeffrey • Lai, Yen-Ting
Assignees
US Department of Health and Human Services
Publication Number
US-11136356-B2
Publication Date
2021-10-05
Expiration Date
2038-10-16
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Abstract
Immunogens comprising a recombinant HIV-1 Env ectodomain trimer stabilized in a prefusion closed conformation and methods of their use and production are disclosed. In several embodiments, the immunogen can be used to elicit an immune response to HIV-1 in a subject.
Core Innovation
Millions of people worldwide are infected with HIV-1, with millions of new infections occurring annually. Despite the availability of effective antiretroviral therapies, over a million people die from AIDS each year, highlighting the need for preventive measures. HIV-1 is an enveloped virus that evades immune recognition through protective mechanisms, with its envelope protein (gp160) cleaved into gp120 and gp41 during infection. The Env spike, composed of gp120 and gp41, is a target for neutralizing antibodies, and an effective immunogen based on HIV-1 Env glycoprotein could elicit a protective neutralizing response. However, agents capable of eliciting such responses remain needed.
This disclosure provides recombinant HIV-1 Env ectodomain trimers stabilized in a prefusion closed conformation through specific amino acid substitutions, which reduce binding affinity for the CD4 receptor while retaining affinity for broadly neutralizing HIV-1 antibodies. Such stabilized trimers include cysteine substitutions forming non-natural intra-protomer disulfide bonds at positions 501 and 605, a proline substitution at position 559, and additional substitutions including methionine at position 302, leucine at position 320, and proline at position 329, according to the HXB2 numbering system. These immunogens, in soluble or membrane-anchored forms, can elicit a neutralizing immune response to HIV-1 when administered to a subject.
The stabilized HIV-1 Env ectodomain trimers demonstrate improved thermostability and reduced recognition by non-neutralizing antibodies targeting CD4-induced epitopes, indicating retention of the vaccine-preferred prefusion closed conformation. The disclosed designs include modifications of the furin cleavage site for enhanced cleavage, as well as glycosylation modifications to influence antibody binding. The recombinant Env trimers can be incorporated into protein nanoparticles, conjugated to carriers, or included in virus-like particles, enhancing their utility as immunogens. Methods for eliciting immune responses, treating, inhibiting or preventing HIV-1 infection in subjects using these stabilized trimers are also provided.
Claims Coverage
The patent claims encompass several inventive features centered on stabilized recombinant HIV-1 Env ectodomain trimers and related embodiments.
Stabilization of HIV-1 Env ectodomain trimer in prefusion closed conformation by specific amino acid substitutions
A recombinant HIV-1 Env ectodomain trimer stabilized by cysteine substitutions at positions 501 and 605 forming a non-natural intra-protomer disulfide bond, a proline substitution at position 559, methionine substitution at position 302, leucine substitution at position 320, and proline substitution at position 329, capable of eliciting an immune response to HIV-1.
Additional disulfide bond stabilization
Inclusion of cysteine substitutions at positions 201 and 433 in protomers forming a non-natural intra-protomer disulfide bond for further stabilization of the trimer.
Specific amino acid substitutions defined by HXB2 numbering
Substitutions including I201C and A433C (cysteine), A501C and T605C (cysteine), I559P (proline), N302M (methionine), T320L (leucine), and A329P (proline), contributing to trimer stability.
Modified furin cleavage site
Substitution of native gp120/gp41 furin cleavage site amino acids with sequences such as RRRRRR (SEQ ID NO: 8), GRRRRRR (SEQ ID NO: 27), GGSGRRRRRR (SEQ ID NO: 28), GRRRRRRRRR (SEQ ID NO: 29), or GNSTHKQLTHHMRRRRRR (SEQ ID NO: 30).
Introduction of N-linked glycosylation site at position 332
Substitutions to introduce an N-linked glycosylation site at position 332 in protomers if not already present to influence antibody binding.
Application across multiple HIV-1 clades
The stabilized recombinant Env trimers encompass sequences from Clade A, B, C, D, or F including the amino acid substitutions for stabilization.
Defined gp120 and gp41 ectodomain boundaries
Protomers comprise gp120 from positions 31-507 and gp41 ectodomain from positions 512-664 according to HXB2 numbering.
Binding affinity constraint to soluble CD4
The recombinant Env ectodomain trimer binds soluble CD4 with affinity tighter than 350 nM, indicating controlled receptor interaction.
Sequence identity and specific sequences
Protomers have at least 90% sequence identity to specified sequences including SEQ ID NOs: 3, 5, 19, 21, 23, 24, or 31-32, or comprise those sequences exactly.
Solubility and trimerization domain linkage
The recombinant HIV-1 Env ectodomain trimer is soluble and optionally linked via peptide linkers or directly to trimerization domains such as T4 fibritin.
Conjugation to heterologous carrier proteins
The trimer can be conjugated to carriers including KLH, CCH, OVA, bovine serum albumin, tetanus toxoid, diphtheria toxoid variants and others to enhance immunogenicity.
Membrane anchoring
Protomers can be linked via peptide linkers or directly to transmembrane domains and may comprise full-length gp41 proteins for membrane anchoring.
Protein nanoparticle formation
Protomers can be linked to subunits of protein nanoparticles such as lumazine synthase, ferritin, or encapsulin to form multimeric nanoparticles displaying Env trimers.
Inclusion in virus-like particles
The recombinant HIV-1 Env ectodomain trimers can be included in virus-like particles for vaccine or immunogenic applications.
Nucleic acid encoding the recombinant trimer
Nucleic acid molecules encoding protomers or protomers linked to nanoparticle subunits, including precursor sequences and embodiments operably linked to promoters, are claimed.
Immunogenic compositions and methods of use
Compositions comprising the recombinant Env ectodomain trimer with pharmaceutically acceptable carriers and optionally adjuvants, and methods of eliciting immune responses or treating HIV-1 infections by administering effective amounts.
The claims define recombinant HIV-1 Env ectodomain trimers stabilized in the prefusion closed conformation by specific amino acid substitutions including cysteine-formed disulfide bonds and proline and hydrophobic substitutions that reduce CD4 binding and retain broad neutralizing antibody recognition. Additional claimed features encompass modifications to furin cleavage sites, glycosylation patterns, trimer solubility, linkage to trimerization domains or carriers, expression constructs, protein nanoparticles, virus-like particles, and immunogenic compositions and methods for eliciting immune responses against HIV-1.
Stated Advantages
Improved thermostability and antigenicity of the HIV-1 Env ectodomain trimers relative to prior forms.
Reduced binding affinity to CD4 while maintaining binding to broadly neutralizing antibodies.
Reduced recognition by non-neutralizing antibodies targeting CD4-induced epitopes.
Suitability for genetic immunization and use as serological probes.
Documented Applications
Use of recombinant HIV-1 Env ectodomain trimers to elicit neutralizing immune responses against HIV-1 in subjects.
Treatment, inhibition, or prevention of HIV-1 infection in subjects using the stabilized Env trimers or nucleic acids encoding them.
Incorporation of the trimers into protein nanoparticles, carrier conjugates, or virus-like particles for vaccine development.
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