Abstract
Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes. Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli. Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10. The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s. Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map. A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity. Antisera against the mobile loop deletion variants exhibited increased cross-reactivity, most especially the antisera against the strongly stabilized variant. The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response.
Highlights
The structure of protein antigens controls the immunogenicity of helper T-cell epitopes at the level of antigen processing as well as binding of peptides to class II MHC1 proteins
Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli
Earlier studies have mapped helper T-cell epitopes to sequences flanking the mobile loop of Mycobacterium leprae Hsp10 in infected humans and immunized mice [5,6,7], but the possibility that the mobile loop should be responsible for the immunodominance of these epitopes has only been hypothesized [1]
Summary
The structure of protein antigens controls the immunogenicity of helper T-cell epitopes at the level of antigen processing as well as binding of peptides to class II MHC1 proteins. As in all Hsp10s examined far, T4Hsp has a flexibly disordered Hsp60-binding mobile loop [3] that we suspected would favor presentation of flanking sequences to helper Tcells. Earlier studies have mapped helper T-cell epitopes to sequences flanking the mobile loop of Mycobacterium leprae Hsp in infected humans and immunized mice [5,6,7], but the possibility that the mobile loop should be responsible for the immunodominance of these epitopes has only been hypothesized [1]. We examined the propensity for three-dimensional structure to influence processing and presentation of T4 Hsp by mapping T-cell epitopes in mice. The immunogenicity of distal epitopes was increased by mobile loop deletions that only modestly affected proteolytic sensitivity. Antibodies raised against the deletion variants exhibited increased cross-reactivity, indicating that antibody epitope immunodominance shifted from epitopes within the loop to epitopes outside of the mobile loop
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