Conformational instability governed by disulfide bonds partitions the dominant from subdominant helper T-cell responses specific for HIV-1 envelope glycoprotein gp120

Abstract

Most individuals infected with human immunodeficiency virus type 1 (HIV-1) generate a CD4+ T-cell response that is dominated by a few epitopes. Immunodominance may be counterproductive because a broad CD4+ T-cell response is associated with reduced viral load. Previous studies indicated that antigen three-dimensional structure controls antigen processing and presentation and therefore CD4+ T-cell epitope dominance. Dominant epitopes occur adjacent to the V1-V2, V3, and V4 loops because proteolytic antigen processing in the loops promotes presentation of adjacent sequences. In this study, three gp120 (strain JR-FL) variants were constructed, in which deletions of single outer-domain disulfide bonds were expected to introduce local conformational flexibility and promote presentation of additional CD4+ T-cell epitopes. Following mucosal immunization of C57BL/6 mice with wild-type or variant gp120 lacking the V3-flanking disulfide bond, the typical pattern of dominant epitopes was observed, suggesting that the disulfide bond posed no barrier to antigen presentation. In mice that lacked gamma interferoninducible lysosomal thioreductase (GILT), proliferative responses to the typically dominant epitopes of gp120 were selectively depressed, and the dominance pattern was rearranged. Deletion of the V3-flanking disulfide bond or one of the V4-flanking disulfide bonds partially restored highly proliferative responses to the typically dominant epitopes. These results reveal an acute dependence of dominant CD4+ T-cell responses on the native gp120 conformation.

Deimmunizing substitutions in Pseudomonas exotoxin domain III perturb antigen processing without eliminating T-cell epitopes

“…3). In contrast, mice immunized with L552E or T18 may not respond to peptide 8 because the epitope is sequestered by the stabilized structure in L552E and T18, as has been described for particular epitopes in other antigens (14,47). Peptide 16 elicited a response from only mice immunized with WT PE-III, and this may be for the same reasons as noted for peptide 8.…”

“…T18 PE-III exhibited no difference in acid-induced unfolding when compared with WT PE-III. Free energies of unfolding were determined by fitting the denaturant-induced unfolding curves as described previously (14,26). The measured free energy of unfolding for T18 PE-III did not differ from that for WT PE-III, whereas the single mutant variants exhibited significant changes in measured free energy compared with WT PE-III ( Fig.…”

“…The presentation of antigen peptides bound to class II MHC molecules depends on proteolytic processing of those antigens. Previous work from our laboratory has demonstrated that the proteolytic processing of antigens preferentially occurs at sites within and between protein domains that are flexible, solventaccessible, and lacking in secondary structure (11)(12)(13)(14)28). These regions are readily accessible for interactions with proteases and can easily conform to protease active sites, making them the initial sites of proteolytic cleavage within an antigen.…”

“…For unfolding free energy determinations, 0.5 M protein was mixed with guanidine-HCl in PBS in 0.25 M steps from 0 to 5 M, and intrinsic tryptophan fluorescence was measured by exciting at 285 nm and scanning emissions from 300 to 400 nm. Fluorescence intensities averaged for the range 330 to 340 nm and for three replicates were analyzed by nonlinear regression as described (14,26). Best-fit values of pH 50 and ⌬G 0 Ј were compared using ANOVA (Prism 6).…”

Exogenous antigens bind MHC class II first, and are processed by cathepsins later

“…Along these thoughts, GILT (gamma interferon-inducible lysosomal thiol reductase) has been shown to enhance antigen processing for antigens with several disulfide bonds such as HEL and HA protein of influenza (Hastings and Cresswell, 2011; Nguyen et al, 2015). The open-ended nature of the MHC class II peptide binding groove supports the ‘binding first, trimming later’ model.…”