David, Chella S.

ABSTRACT A subunit vaccine for Plasmodium falciparum malaria will need to contain well-defined T helper cell epitopes that induce protective immune responses to the parasite. One major barrier to the use of subunit vaccines is the requirement for T helper cell epitopes to be presented by the HLA class II molecules that are present in the population being vaccinated. Since the majority of malaria studies have focused on HLA-DR, little information on the role of HLA-DQ in the binding and immune response to malarial epitopes is available. This study used an in vitro peptide-binding assay to predict the extent of HLA-DQ binding of four conserved T helper cell epitopes identified from asexual-stage malaria vaccine candidate antigens. Epstein-Barr virus (EBV)-transformed human B-cell lines expressing 14 different DQ molecules (DQ2.1, -2.2, -4.1, -4.2, -5.1 to -5.3, -6.1, -6.2, -6.4, -7.1, -7.3, -8, and -9) representing all broad serological specificities, including common DQ molecules present in populations in areas where malaria is endemic, were used in the binding assay. Moreover, an HLA-DQ transgenic mouse model was employed to evaluate the correlation between the in vitro DQ binding of the peptides and the generation of in vivo immune responses following peptide immunization. This study identified two broad DQ-binding peptides, ABRA#14 and SERA#9. ABRA#14 also induced T-cell proliferation and Th1-associated cytokine production in DQ8 + transgenic mice. The combination of peptide binding to EBV-transformed cell lines and DQ transgenic mice provides a method for identifying additional T-cell epitopes for inclusion in a vaccine.

Although DQA1*0301/DQB1*0302 is the human histocompatibility leukocyte antigen (HLA) class II gene most commonly associated with human type 1 diabetes, direct in vivo experimental evidence for its diabetogenic role is lacking. Therefore, we generated C57BL/6 transgenic mice that bear this molecule and do not express mouse major histocompatibility complex (MHC) class II molecules (DQ8+/mII−). They did not develop insulitis or spontaneous diabetes. However, when DQ8+/mII− mice were bred with C57BL/6 mice expressing costimulatory molecule B7-1 on β cells (which normally do not develop diabetes), 81% of the DQ8+/mII−/B7-1+ mice developed spontaneous diabetes. The diabetes was accompanied by severe insulitis composed of both T cells (CD4+ and CD8+) and B cells. T cells from the diabetic mice secreted large amounts of interferon γ, but not interleukin 4, in response to DQ8+ islets and the putative islet autoantigens, insulin and glutamic acid decarboxylase (GAD). Diabetes could also be adoptively transferred to irradiated nondiabetic DQ8+/mII−/B7-1+ mice. In striking contrast, none of the transgenic mice in which the diabetes protective allele (DQA1*0103/DQB1*0601, DQ6 for short) was substituted for mouse MHC class II molecules but remained for the expression of B7-1 on pancreatic β cells (DQ6+/mII−/B7-1+) developed diabetes. Only 7% of DQ−/mII−/B7-1+ mice developed diabetes at an older age, and none of the DQ−/mII+/B7-1+ mice or DQ8+/mII+/B7-1+ mice developed diabetes. In conclusion, substitution of HLA-DQA1*0301/DQB1*0302, but not HLA-DQA1*0103/DQB1*0601, for murine MHC class II provokes autoimmune diabetes in non–diabetes-prone rat insulin promoter (RIP).B7-1 C57BL/6 mice. Our data provide direct in vivo evidence for the diabetogenic effect of this human MHC class II molecule and a unique “humanized” animal model of spontaneous diabetes.

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4 + human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab 0 ) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4 + T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis , and only a single hsp65 epitope, p1–20. DR3.Ab 0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis , and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab 0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab 0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab 0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab 0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis . Thus, for two different Ags, T cells from DR3.Ab 0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.