Envelope Deglycosylation Enhances Antigenicity of HIV-1 gp41 Epitopes for Both Broad Neutralizing Antibodies and Their Unmutated Ancestor Antibodies

Abstract

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…In general, our findings are consistent with a recent report on PNGase F-mediated deglycosylation of uncleaved JR-FL and CON-S gp140 trimers (43). Thus, in both studies, a subset of the total glycans could not be removed.…”

“…For example, the asparagine at gp41 position 637 was not occupied on the uncleaved JR-FL and CON-S gp140 trimers, but is used on the cleaved KNH1144 trimers. In addition, we found that the glycans at positions 301 and 448 were removed, whereas they were not efficiently digested away from the uncleaved trimers (43). Whether differences in the design of the trimers (cleaved versus uncleaved, with or without the gp41 fusion peptide), the HIV-1 sequences on which they are based (KNH1144 versus JR-FL/CON-S), the deglycosylation protocol (Endo H versus PNGase F), or the analytical method used to assess deglycosylation, underlie these differences remains unknown.…”

“…Deglycosylating trimeric Env on virions has proven problematic, probably because steric factors restrict access of glycosidases to their substrates (17,42). Partially deglycosylated, uncleaved gp140 trimers have recently been described (43).…”

Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

“…A strategy for designing such an immunogen, on the basis of analysis of both the structure and the lineage, could include (in addition to the native HA) a component to induce a UCA-like primary response (17). Inspection of the differences between CH65 and its UCA suggests that the principal changes affecting affinity are in the light-chain CDR1, where mutations at positions 26 and 29 have introduced salt bridges with HA (Table S4).…”

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

“…Adding to the challenge has been the difficulty in achieving binding of proposed antigens to germ-line or unmutated common ancestors (UCAs). Binding to BnAb UCAs would be a desirable characteristic for putative immunogens intended to induce BnAbs (5,(9)(10)(11)(12)(13).…”