Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.

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We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.

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In this study we demonstrate that immunization of H-2(b) mice with the allergen Der p 1 induces MHC class II restricted T cells that proliferate to residues 15-29 of Der p 1 (p15-29) and to the murine MHC class II-associated invariant chain derived peptide (CLIP). T cells from naive H-2(b) mice and those immunized with murine CLIP fail to respond to either CLIP or p15-29. T cell lines and clones reactive with p15-29 strongly proliferate in response to splenic antigen-presenting cells (APC) from normal H-2(b) mice but show reduced proliferation to APC from invariant chain deficient mice. Furthermore, T cells isolated from Der p 1 primed mice and expanded on H-2(b) spleen cells in the absence of the p15-29 epitope retained specificity for both p15-29 and CLIP, suggesting that naturally presented self components can act as mimetic peptides and may maintain T cell memory to foreign antigens.

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