Characterization of Human Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120 Glycoproteins Stabilized in the CD4-Bound State: Antigenicity, Biophysics, and Immunogenicity

Abstract

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S؉S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S؉S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

“…The majority of immunogens studied to date elicit sera that neutralize a subset of Tier 1 viruses but fail to neutralize most Tier 2 and 3 viruses. Consistent with earlier studies (18,19), sera from the control group largely neutralized Tier-1 neutralization-sensitive viruses. Depletion studies and competition binding assays with b12 showed that the antibodies in the broadly neutralizing sera are gp120-directed, and an appreciable fraction of antibodies in group 3 sera is directed toward the CD4 binding site.…”

Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens

“…Serum from macaque 841, which neutralized the virus in the absence of sCD4, showed only a modest increase in potency in the presence of sCD4. Of note, this variable pattern of neutralization among the rhFLSCimmunized macaques resembles what was measured in a similar assay of rabbit sera raised against stabilized envelope trimers that present CD4-induced epitopes (30). In comparison, serum from only one animal in the gp120-CD4 XL group (macaque 842) exhibited neutralizing activity in the presence of sCD4 (Fig.…”

CD4 mimetics sensitize HIV-1-infected cells to ADCC

“…The resistance of most primary HIV-1 Envs to sCD4 engagement may explain why sCD4 exhibited only minimal in vivo potential as an antiviral agent (44). CD4mc have been shown to sensitize HIV-1 virions to neutralization by otherwise nonneutralizing CD4i Abs that can easily be generated through vaccination with CD4-bound stabilized gp120s (25,45,46). Importantly, these types of Abs were isolated from RV144 vaccinees and possessed ADCC potential (10,12).…”

“…Therefore, the dual effect of these CD4mc (i.e., sensitization of HIV-1 viral particles to neutralization and infected cells to ADCC by CD4i Abs) might help limit viral transmission. Such CD4i Abs are elicited early and in a high proportion of infected individuals (11,17) and can also be elicited by vaccination with "stabilized gp120 cores" that have been engineered to assume the CD4-bound state (45). One can envision a multicomponent vaccine regimen in which one of the immunogens is a stabilized gp120 core that elicits CD4i Abs and CD4mc administered orally or in a microbicide formulation that could sensitize a range of T/F viruses and infected cells to inhibition by the vaccine-elicited Abs.…”