ENVolution: Immunoselection of Novel HIV Envelope Immunogens for HIV Vaccine Development.

Abstract

A major challenge for human immunodeficiency virus (HIV) vaccine development is the design of HIV envelope (Env) immunogens that elicit broadly neutralizing antibodies (BnAbs). Most of the characterized BnAbs against HIV-1 block the infectivity of a wide spectrum of isolates by targeting conserved regions located on the functional Env trimer such as the CD4-binding site. Vesicular stomatitis virus (VSV), the prototypic member of the Rhabdoviridae family of negative-strand RNA viruses, is a promising vaccine delivery vector because it can infect human cells, but is not a natural human pathogen, has low seroprevalence in most human populations, propagates robustly in culture, induces strong immune responses in vivo and tolerates foreign gene insertion including HIV Env. As with most RNA viruses, the VSV RNA-dependent RNA polymerase lacks proof-reading function and the genome randomly incorporates mutations during replication. Viral variants accumulate during propagation, and evolution of the viral population can be directed by applying selective pressure. For example, recombinant VSV (rVSV) vectors encoding HIV-1 Env in place of its natural attachment and entry protein, VSV G, are under selective pressure to maintain the foreign gene and express functional trimers that specifically infect CD4/CCR5(+) cells. We hypothesized that the application of BnAb-specific selective pressure during serial passage of rVSV-HIV Env vectors could be exploited to generate novel functional Env trimer immunogens. To explore the use of VSV’s evolutionary potential to generate novel HIV-1 Env immunogens, rVSV vectors expressing clade B and clade C Env were constructed and methods to apply selective pressure were developed. First, as proof of concept for the ability of rVSV to rapidly adapt in the presence of a BnAb-directed selective pressure, rVSV viruses expressing HIV-1 Env proteins in place of VSV G were passaged on CD4/CCR5(+) cells in the presence of neutralizing concentrations of b12, a BnAb targeting an epitope that overlaps with the CD4 binding site (CD4s). Four b12-escape variants containing substitutions in the CD4bs of HIV-1 Env were identified: P369L, P369L_V372A, G366D and G366S_T278A. All four variants exhibited decreased binding to b12 and retained CD4-dependent entry despite the presence of substitutions of CD4 contact residues on the Env protein. Only one of the four variants, G366D, was completely resistant to b12 neutralization. The P369L_V372A and G366D variants were 2-fold more resistant to neutralization by the CD4bs-directed BnAb PGV04, but only the G366D variant showed decreased binding to PGV04. Surprisingly, all four variants exhibited an increase in both binding and neutralization by a glycan-specific BnAb, PGT126, which targets a quaternary epitope located on the V3 loop of gp120. Most importantly, these studies for the first time showed the neutralization phenotype of viruses expressing HIV-1 Env with substitutions at residue G366, a residue that is 99% conserved among all circulating HIV-1 isolates and is critical for HIV-1 viability. Second, a positive selection methodology coupled with serial passage was developed to generate novel Env immunogens that potentially have higher binding affinities for BnAbs. The BnAb b12 was used to select for viral variants that maintain binding to b12 under stringent conditions. Two Env variants, N280S and Q652R, were isolated and contained substitutions of residues that are 99% conserved among circulating HIV-1 isolates. The N280S variant contained a substitution in gp120 that is a contact point for b12 and CD4. The Q652R variant contained a substitution in gp41. Both variants retained CD4-dependent entry. b12 binding and neutralization to the N280S variant was unaltered compared to WT. While b12 binding to the Q652R variant was unaltered, this variant was slightly resistant to neutralization by b12. Although both N280S and Q652R variants were more resistant to neutralization by the CD4bs-directed BnAbs VRC01 and PGV04, only variant N280S demonstrated decreased binding to VRC01 and PGV04. Importantly, these studies showed for the first time the neutralization phenotype of viruses expressing HIV-1 Env substitutions at residue N280, a residue that is reported to be critical for HIV-1 viability. These studies show that novel HIV-1 Env proteins can be rapidly and specifically generated by directing the evolution of VSV-HIV EnvG vectors via the application of BnAb-specific selective pressure. By employing this biological system with a panel of BnAbs, the strategies we describe here can complement standard approaches to identify BnAb epitopes, to determine structural conformations required for epitope exposure and provide an improved understanding of BnAb binding and neutralization properties that may lead to the design of improved immunogens. Ultimately, we envision that this system may be used to generate novel Env immunogens as promising candidates for HIV vaccine trials and can be broadly applied to generate novel proteins as promising candidates for other vaccine immunogens or as novel therapeutical agents against disease.