Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at Its Apex

Abstract

The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but they did not always overlap, indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex. The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside V2 increased V2 bnAb recognition, they often also increased Env stability-of-function and decreased binding by narrowly neutralizing antibodies to the V3 crown. Three mutations together increased neutralization by V2 bnAb and eliminated binding by V3 crown antibodies. These results may aid the design of immunogens that elicit antibodies to the trimer apex.

A rare mutation in an infant derived HIV-1 envelope glycoprotein alters interprotomer stability and susceptibility to broadly neutralizing antibodies targeting the trimer apex

“…As V1V2 stabilizes the Env spike forming the trimer apex, we next performed a stability-of-function assay, called T90 assay, that determines Env stabilization (viral infectivity) as a function of temperature (38, 39), to evaluate the effect of the L184F mutation on the Env stability. A slight increase in T90 value, the temperature at which the viral infectivity decreased by 90% in 1 hour, from 43.27 to 43.69 (p = 0.19) was observed, though the impact of L184F on thermal stability did not appear markedly noticeable ( data not shown ).…”

“…Changes at the trimer apex have been shown to alter virus sensitivity and often come with a fitness cost (39–42). In order to investigate the effect of acquisition of the rare L184F viral immunotype on the functional stability of the Env, we assessed the impact of L184F mutation on viral infectivity in free virus and cell-cell transmission.…”

“…As V2-apex bnAbs target the closed conformation of the trimer, based on neutralization profile observed, we hypothesized that the most plausible reason for the loss of susceptibility to V2-apex bnAbs was the loss of closed conformation of the Env. In its closed conformation, the Env trimer occludes several immunodominant epitopes that are targeted by non-neutralizing antibodies (non-nAbs) (39, 41). When neutralization profile against several non-nAbs (those targeting CD4-induced epitopes and V3 loop) was assessed, the L184F mutant virus showed relatively higher neutralization susceptibility to non-nAbs.…”

“…Substitution of the small side chain of leucine with the bulky non-polar side chain of phenylalanine led to disruption of interprotomer interactions that have been observed to be critical for maintenance of the trimeric apex. Mutations that alter or disrupt interprotomer contacts in Env trimer have been shown to change its susceptibility to several classes of bnAbs (39, 41–43). Overall, our results provide information on the role of a rare escape mutation L184F in the viral envelope that led to resistance to bnAbs targeting the V2-apex.…”

“…Thermostability (T90) assays were performed as described previously (38, 39). Briefly, 73105b and 73106f pseudotyped viruses were incubated at temperatures from 37°C to 50°C for 60 minutes using temperature gradient on PCR thermal cycler (BioRad).…”