Structure-Function Studies of the HIV-1 Envelope Glycoproteins and Development of VLP-forming vaccine strategies for HIV-1

National Institute Of Allergy And Infectious Diseases

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Abstract

Although a protective HIV vaccine may provide one of the most effective means to terminate the HIV/AIDS pandemic, its development has been delayed and hampered by unprecedented challenges, primarily as a consequence of the unique properties of the HIV-1 envelope (Env) trimer, which features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and conformational masking of key antigenic sites targeted by neutralizing antibodies. Further insights into the complex structure-function relationships within the HIV-1 Env trimer may be critical to inform the rational design of a protective vaccine and other immunoprophylactic measures. 1) Enhancement of broadly HIV-neutralizing antibodies. Passive transfer of bNAbs is a promising alternative to ART for HIV-1 prevention and treatment. We previously reported the discovery of a second CD4-binding site in the HIV-1 Env trimer, that we defined as CD4-binding site 2 (CD4-BS2), which opened new perspectives for HIV vaccine and therapy (Liu et al., NSMB 2018). We also rationally engrafted the extended FR3 loop of VRC03 onto different anti-CD4-supersite bNAbs and developed chimeric antibodies with enhanced activity against a wide panel of global HIV-1 strains (Liu et al., 2019, 2021a and 2021b). During this year, we applied this strategy to a new family of anti-CD4bs bNAbs (i.e., the N49 lineage) and found that the resultant chimeric antibodies bind the Env trimer with a higher affinity than their parental forms and display increased neutralizing activity against a global panel of HIV-1 Envs. In particular, through a series of rational modifications, we produced a variant, N49P9.6-FR-LS, that demonstrates enhanced potency, superior antiviral activity in combination with other bnAbs, low polyreactivity, and longer circulating half-life. Additional engineering for manufacturing produced a final variant, eN49P9, with properties conducive to cGMP production (Sajadi et al., 2025). Due to their increased potency associated with reduced autoreactivity and prolonged in vivo half-life, FR3-loop-chimeric bNAbs are under consideration for clinical use in HIV-1 prevention and treatment. In parallel studies, we are also trying to design more potent chimeric antibodies by screening recombinant libraries of FR3 segments to be inserted into the bNAb VRC07. 2) Design of soluble HIV-1 envelope trimers free of covalent gp120-gp41 bonds with prevalent native-like conformation. Soluble HIV-1 envelope trimers may serve as effective vaccine immunogens. The widely utilized SOSIP trimers have been paramount for structural studies, but the disulfide bond they feature between gp120 and gp41 constrains intersubunit mobility and may alter antigenicity. We developed an alternative strategy to generate stabilized soluble Env trimers free of covalent gp120-gp41 bonds (Zhang et al., 2024). Stabilization was achieved by introducing an intrasubunit disulfide bond between the inner and outer domains of gp120, defined as interdomain lock (IDL). Correctly folded IDL trimers displaying a native-like antigenic profile were produced for HIV-1 Envs of different clades. Importantly, the IDL design abrogated CD4 binding while not affecting recognition by potent neutralizing antibodies to the CD4-binding site. By cryoelectron microscopy, IDL trimers were shown to adopt a closed prefusion configuration, while single-molecule fluorescence resonance energy transfer documented a high prevalence of native-like conformation. Thus, IDL trimers may be promising candidates as vaccine immunogens. 3) Characterization of a quaternary-binding broadly neutralizing antibody and its ontogeny. Although most bNAbs specific for the CD4-binding site (CD4-BS) of HIV-1 interact with a single gp120 protomer, a few mimic the quaternary binding mode of CD4. This year, we identified a CDRH3-dominated anti-CD4-BS bNAb, CH103, which establishes quaternary interaction despite possessing a regular-length FRH3 and CDRH1 (Liu et al., 2025). We demonstrated that such quaternary interaction is critical for neutralization and is primarily mediated by two FRH3 acidic residues that were sequentially acquired and subjected to strong positive selection during CH103 maturation. Cryoelectron microscopy (cryo-EM) structures confirmed the role of the two FRH3 acidic residues in mediating quaternary contact and demonstrated that CH103 reaches the adjacent gp120 protomer by virtue of its unique angle of approach. Thus, the acquisition of quaternary interaction may constitute a key step in the lineage maturation of a broad and potent HIV-1 neutralizing antibody. 4) Improvement of a VLP-forming vaccine against HIV-1 by inclusion of a retroviral protease. To improve the efficacy of our VLP-forming vaccine platform, we introduced a key technological advance by inclusion of a retroviral protease to process Gag and produce mature VLPs (Zhang et al., 2025). Appropriately dosed and timed expression of the protease was achieved using a full-length gag-pol mRNA transcript. Inclusion of the protease resulted in enhanced titers of trimer-binding and neutralizing antibodies in a mouse model. Analogous results were obtained using a hybrid Gag-based, VLP-forming severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine expressing an engineered spike protein. Thus, inclusion of a retroviral protease can increase the immunogenicity of Gag-based, VLP-forming vaccines against different human pathogens.