We have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class IL In this study,... Show moreWe have reported that the major histocompatibility molecule HLA-DQ2 (DQA1*05:01/DQB1*02:01) (DQ2) is relatively resistant to HLA-DM (DM), a peptide exchange catalyst for MHC class IL In this study, we analyzed the role of DQ2/DM interaction in the generation of DQ2-restricted gliadin epitopes, relevant to celiac disease, or DQ2-restricted viral epitopes, relevant to host defense. We used paired human APC, differing in DM expression (DMnull versus DMhigh) or differing by expression of wild-type DQ2, versus a DM-susceptible, DQ2 point mutant DQ2 alpha+53G. The APC pairs were compared for their ability to stimulate human CD4(+) T cell clones. Despite higher DQ2 levels, DMhigh APC attenuated T cell responses compared with DMnull APC after intracellular generation of four tested gliadin epitopes. DMhigh APC expressing the DQ2 alpha+53G mutant further suppressed these gliadin-mediated responses. The gliadin epitopes were found to have moderate affinity for DQ2, and even lower affinity for the DQ2 mutant, consistent with DM suppression of their presentation. In contrast, DMhigh APC significantly promoted the presentation of DQ2-restricted epitopes derived intracellularly from inactivated HSV type 2, influenza hemagglutinin, and human papillomavirus E7 protein. When extracellular peptide epitopes were used as Ag, the DQ2 surface levels and peptide affinity were the major regulators of T cell responses. The differential effect of DM on stimulation of the two groups of T cell clones implies differences in DQ2 presentation pathways associated with nonpathogen- and pathogen-derived Ags in vivo. Show less
Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses... Show moreAttenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines. Show less
