Comprehensive analysis of contributions from protein conformational stability and major histocompatibility complex class II-peptide binding affinity to CD4+ epitope immunogenicity in HIV-1 envelope glycoprotein

Abstract

Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing.

Importance: CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an HIV protein partially determines which epitopes are dominant, most likely by controlling the breakdown of HIV into peptides. Moreover, some types of signals from CD4+ T cells are affected by the HIV protein 3D structure; and thus the protectiveness of a particular peptide vaccine could be related to its location in the 3D structure.

FIG 1

HIV gp120 structure and epitope dominance. (A) The line graph indicates the number of mice responding to a given peptide. The area graph indicates the likelihood that the peptide contains the N-terminal end of a C-terminal proteolytic fragment (L_C-frag). The positions of flexible loops and disulfide bonds are indicated above the graph. Disulfide bonds that were individually deleted in gp120 variants are indicated with heavy brackets, under which the position of the N-terminal cysteine is given. The bar graph indicates the I-A^k peptide-binding motif score (see the text). The six dominant epitopes were positive for an IL-2 response in three or more mice. Each dominant epitope lies on the C-terminal flank of a conformationally unstable loop. Peptide 30 was the only peptide that contained both a dominant epitope and an I-A^k-binding motif. (B) Relationship between conformational stability and L_C-frag in the V3 loop and neighboring sequence positions. The aggregate conformational stability score is shown by the thin line; any residue with a score above zero is considered to be part of a stable segment. In the stable segments, L_C-frag (thick line) is equal to the aggregate conformational stability with weighting to emphasize epitope probability at the N termini, such that the conformational stability score is upweighted 3-fold for the N-terminal 24 residues of the stable segment and downweighted 3-fold for the C-terminal 24 residues. Additionally, the flexible region preceding the stable segment is increased linearly from zero at the midpoint of the flexible segment to the upweighted peak of the stable segment. (C) Ribbon diagrams of gp120 illustrating positions of the conformationally unstable segments in gp120.

FIG 2

Enhanced T-cell responses for disulfide variants of gp120. (A) Each symbol is the average response to 38 individually tested peptides for splenocytes of a single mouse. Bars and asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Average peptide-specific IL-4 responses following immunization of nine mice with wild-type gp120 and disulfide variants. (C) Differences in average secreted IL-4 for disulfide variants versus wild-type gp120. The dashed horizontal line indicates the average difference for all peptides. Bar graphs indicate the I-A^k motif score. I-A^k+ peptides were associated with above-average differences in IL-4 secretion, i.e., were preferentially affected in gp120dss298 and gp120dss385. For the most destabilized disulfide variant, gp120dss298, 12 of 14 I-A^k+ peptides were preferentially affected, and the two exceptions are peptides 30 and 38, which are located in flexible loops.

FIG 3

Th1-skewed responses induced by addition of CpG oligonucleotide with mLT as an adjuvant for immunization with gp120. (A) Each symbol is the average response to 38 individually tested peptides for splenocytes of a single mouse. Bars indicate significant differences between mLT and mLT+CpG as in the legend for Fig. 2. (B) Peptide-specific IFN-γ responses following immunization with gp120 and the indicated adjuvant formulation. (C) Difference in secreted IFN-γ for mLT+CpG versus mLT alone. The dashed horizontal line indicates the average difference for all peptides. The bar graph indicates the I-A^k motif score. Immunization of mice with gp120 and mLT or mLT+CpG produced large T-cell responses. Comparing mLT+CpG and mLT, the responses for mLT+CpG were skewed toward Th1, as indicated by the elevated IFN-γ and diminished IL-4, IL-5, and IL-6 amounts. Increases in IFN-γ were not preferentially associated with I-A^k+ peptides; rather, they followed the original dominance pattern.

FIG 4

Processing and presentation of a dominant epitope and an I-A^k+ epitope that is concealed by a disulfide bond. Proteolytic cleavage makes the dominant epitope available for loading into the MHC protein. In the case of an intact disulfide bond (upper pathway), the I-A^k+ epitope remains unavailable for loading because the disulfide bond stabilizes the partially folded conformation. In the case of a mutated disulfide bond (lower pathway), the I-A^k+ epitope is presented because the protein undergoes additional unfolding.