Elicitation of HIV-specific Antibodies Targeting The Carbohydrate-Occluded Neutralization Epitopes Through Rational Protein Design

Background The HIV‐1 viral surface Env protein encodes a dense array of carbohydrates. Many of these N‐linked surface‐covering carbohydrates appear at highly conserved positions, although most HIV‐1 isolates are missing one or more glycosylation sites, which potentially reveal underlying surface structures. We have grouped the Env surface that underlies these carbohydrates into a set of Carbohydrate‐Occluded Neutralization Epitopes (CONEs). We have examined the utility of small protein mimics of the CONEs to serve as potential immunogens. Methods We selected four distinct CONEs as targets (three‐stranded β‐sheet, α‐helix, and two separate loops) and identified compatible motifs in the PDB for each epitope‐scaffold design. We then grafted the exposed residues of each CONE onto the respective scaffolds followed by computational redesign to increase thermodynamic stability. The chimeric proteins were expressed in E. coli and purified then used in structural analysis and rabbit immunization. Results We determined the structures of two designed proteins C2S5 and C4S3 as mimetics of the α2‐helix and the C loop of Env. Crystal structures were solved to resolutions of 1.2 Å and 2.0 Å, respectively. Comparison with the SOSIP trimer structure (PDB 4TYP) revealed a high degree of mimicry: within the epitope region the overall RMSDs between the designs and Env structure are 0.64 Å (C2S5) and 0.36 Å (C4S3), suggesting high‐accuracy structural design and our accurate presentation of the CONE residues on heterologous protein scaffolds. In addition to the primary scaffolds we also made alternative scaffolds to examine the specificity of the corresponding antibodies. We generated antibodies to the primary immunogens and detected cross‐reactivity to the alternative scaffolds by ELISA, suggesting the antibodies react to the HIV‐specific segment of our immunogens. We next evaluated the interactions between the polyclonal antibodies and gp120 (fully‐glycosylated and under‐glycosylated) through western blot. The rabbit sera reacted specifically to the under‐glycosylated gp120. Importantly, we observed antibody‐mediated neutralization towards tier 2 viruses with under‐glycosylated Env. Conclusions We have been able to design structural mimetics of surface features of the HIV‐1 Env protein that are typically but not always occluded by carbohydrate, and to use these mimetics to raise HIV‐specific antibodies. These small and thermally stable immunogens represent a starting point for eliciting a new class of antibodies that in combination could neutralize most isolates. Support or Funding Information NIH Grant R01AI102732