(649e) Exploring the HIV V1V2 Loop Conformational Landscape with Protein Engineering

Developing an effective HIV vaccine remains an elusive goal; the vast sequence diversity of HIV presents an enormous challenge with respect to identifying vaccine targets for broad protection. Characterization of several classes of broadly neutralizing antibodies isolated from infected individuals has identified several ‘sites of vulnerability’ on the HIV envelope protein such as the V1V2 loops, and vaccine-induced antibody responses to these sites may provide protection. Structure-based vaccine design approaches have sought to utilize structural understanding of these antibodies’ recognition of their epitopes to inform design of epitope-focusing immunogens. Despite advances in structural knowledge with the crystallization of the full-length HIV envelope trimer, our limited understanding of the more conformationally dynamic V1V2 loops may hinder structure-based immunogen design for this site.

The V1V2 loops had previously been considered as intractable targets for vaccine design because of their sequence variation, yet their structural and functional conservation, their association with vaccine-mediated protection in the RV144 trial, and the isolation of V1V2 loop-specific broadly neutralizing antibodies now point to their relevance to vaccine design. Monoclonal antibodies recognizing distinct conformations of the V1V2 loops suggest that these loops may exist dynamically, and may be presented differentially by monomeric V1V2 and trimeric envelope spikes. That antibodies preferring quaternary and conformational epitopes (i.e. PG9 and 697-30D) also exhibit greater neutralization potency and breadth as compared to antibodies recognizing linear epitopes (i.e. CH58 and CH59) suggests that the former may be more desirable in a vaccination setting. Thus, achieving proper conformational presentation of the V1V2 loops in an epitope-focusing immunogen presents a protein engineering problem. Here, we explore the conformational landscape of the V1V2 loops by characterizing the conformational effects of a novel mutation identified by protein engineering.

Through computational design and directed evolution of a mini-V1V2 antigen, we have identified a novel mutation in the V1V2 loops that may result in a more preferable quaternary-like conformation; inclusion of this substitution in our mini-V1V2 antigen design and in monomeric gp70 V1V2 scaffolds results in enhanced binding to antibodies preferring quaternary and conformational epitopes (PG9, PG16, 697-30D), as well as a reduction in binding to antibodies recognizing linear epitopes (CH58 and CH59) in both yeast and mammalian expression systems. The location of this substitution is distal from known anti-V1V2 epitopes and in a region of the V1V2 loops well conserved in sequence and in structure. Additionally, its effect on several classes of antibodies with distinct binding modes, as well as its phenotypic effect across 4 HIV strains covering 3 clades, suggests that a more global structural modification or stabilization of V1V2 secondary structure may be taking place. As a single point mutation, this substitution may have utility in simplifying minimal V1V2 immunogen design, in which current approaches require many mutations or loop grafting for proper V1V2 conformation. Furthermore, inclusion of this substitution in a soluble V1V2 probe may allow for profiling of anti-V1V2 responses with finer resolution towards conformation-specific responses currently missed by existing state-of-the-art probes. Taken together, this result has potential to further elucidate structural intricacies of the V1V2 loops and corresponding antibody responses, which will enhance future structure-based vaccine design efforts.