N. Kalaya Steede

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

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 interferon-inducible 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.

Shaping T cell - B cell collaboration in the response to human immunodeficiency virus type 1 envelope glycoprotein gp120 by peptide priming.

Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.

Domain-specific spectroscopy of 5-hydroxytryptophan-containing variants of Escherichia coli DnaJ.

Tryptophan-containing variants of Escherichia coli DnaJ protein were constructed in order to examine the hypothetical domain structure by fluorescence quenching and denaturant-induced unfolding. Two residues in the J-domain and one in the Gly/Phe-rich region were targeted for replacement and the proteins were expressed in a tryptophan auxotrophic strain in the presence of 5-hydroxytryptophan (5-HW). Fluorescence quenching with iodide of 5-HW in the variant proteins suggests that the Gly/Phe-rich region is more accessible to solvent than the J-domain. This is consistent with the proposal that the Gly/Phe-rich region is unstructured. Unfolding of the 5-HW-containing variants was monitored by fluorescence, and the results showed that the unfolding of the J-domain is cooperative and the unfolding of the Gly/Phe-rich region is not cooperative.