A major challenge in HIV-1 vaccine design is to generate antibodies directed toward conserved broadly neutralizing epitopes on the surface-exposed viral envelope glycoprotein (Env). Most conserved epitopes are masked by self N-glycans, limiting naive B cell recognition of the underlying protein surface following Env vaccination or during natural infection. Recently, soluble faithful mimics of the HIV Env spike have been developed, including the stabilized cleavage-independent NFL (native flexibly linked) trimers, but their capacity to elicit broadly cross-reactive tier 2 (clinical isolate) neutralizing responses has so far been limited. The conserved primary receptor, CD4 binding site, is a known neutralizing determinant, but is flanked by self-N-linked glycans, limiting B cell and antibody access to this site. Here, we eliminated up to 4 N-glycans surrounding the CD4 binding site of the NFL trimer without affecting trimer stability or conformation, as demonstrated by multiple biophysical methods and EM. Using these well-ordered trimers, we arrayed them at high density on synthetic liposomal nanoparticles because, as we have shown, this multivalent trimer array enhances B cell activation, germinal center formation and the elicitation of HIV neutralizing antibodies. We performed immunogenicity experiments in animal models, demonstrating that the N-glycan-deleted trimers elicited superior neutralizing responses as a prime for fully glycosylated trimers, compared to multiple immunizations with fully glycosylated trimers alone. The N-glycan deleted priming also resulted in detectable cross-neutralization of a small subset of tier 2-like viruses following boosting. The approach of N-glycan deletion as a prime, coupled with multivalent trimer array, is a promising means to elicit better HIV cross-neutralizing antibodies.
IAVI Neutralizing Antibody Center at Scripps