Original contributionInduction of CTL response by a minimal epitope vaccine in HLA A∗0201/DR1 transgenic mice: dependence on HLA class II restricted TH response☆
Introduction
During the past decade, considerable interest has been focused on specifically inducing CTL to prevent and/or control chronic viral diseases. Among the numerous approaches investigated, are epitope based studies using synthetic peptides derived from the processed form of viral proteins which bind to MHC class I alleles 1, 2, 3, 4. Protective antiviral immunity using CTL epitope peptides has been achieved in several experimental models of infection 5, 6, 7, 8, 9. The epitope-based approach offers several potential advantages. Selection of naturally processed epitopes may allow the immune system to focus on highly conserved and immunodominant epitopes of a pathogen [10]. The epitope-based approach may also augment CD8+ CTL responses with the inclusion of high potency CD4+ TH (helper T lymphocyte) epitopes into the vaccine construct to sustain antiviral CD8+ T-cell activity 11, 12.
Several investigators have examined the potential of using TT (tetanus toxin) derived peptides to induce strong TH responses, to strengthen the potency of vaccine preparations 8, 13, 14. A large proportion of the population has previously been vaccinated with tetanus toxoid vaccine preparations, therefore, most subjects have TT responsive precursor cells. Because a need for potent TH responses to maximize the duration of activity of adoptively transferred CTL or peptide vaccines has been shown, it would be of great value to harness the immune-stimulating capacity of tetanus vaccinations 15, 16. With the aid of the murine transgenic model discussed below, immunogenic determinants from TT protein, which bind to multiple HLA DR alleles 17, 18, 19, 20, 21 have been evaluated for optimal induction of HLA-A∗0201-restricted and CMV-specific CTL responses in vivo.
As a preclinical model, a transgenic (Tg) mouse that expresses both HLA-A∗0201 class I and HLA-DR1 class II molecules was established. The HLA A∗0201 transgene used in these studies encodes a chimeric molecule in which the α3 domain of the HLA-A∗0201 heavy chain was replaced with the corresponding domain from the murine H-2 Kb heavy chain [22]. The HLA-DR1 transgene is a chimeric (human/mouse) molecule (DRB1∗0101, DRA1∗0101) in which the α2 domain of the HLA-DR1 α chain was replaced with the corresponding domain from the murine I-Eα chain, thereby enhancing interaction with murine CD4 [23]. This was done since, at least for some HLA class II molecules, there is a species barrier to the interaction with murine CD4 [24]. The model has been used to test the role of HLA class II in the induction and potency of HLA class I restricted CTL responses against a CMV-specific CTL epitope.
We report that both HLA-A∗0201 and HLA-DR1 transgenes are functional in vivo as MHC class I and class II restriction elements, respectively, and that the product of the HLA-DR1 transgene enhances HLA-A∗0201-restricted CMV-specific CTL responses. Of particular interest is the fact that a single dose of the HLA-A∗0201 restricted pp65495–503 CTL epitope, in combination with three different TT derived TH epitopes; TT639–652, TT830–843, and TT947–967, resulted in the induction of a vigorous CMV-specific CTL response in HLA-A∗0201/DR1 Tg mice. These results have both theoretical and practical importance for the development of prophylactic and therapeutic peptide vaccines against pathogens.
Section snippets
Generation of Tg mice expressing HLA-DR1 and HLA-A∗0201 molecules
The HLA-A∗0201 transgene is a chimeric molecule consisting of the α1 and α2 domains of the HLA-A∗0201 molecule and the α3 domain of murine H-2Kb 22, 25. The substitution of the α3 domain of the murine Kb molecule is required to strengthen the interactions with endogenous murine CD8, producing a more vigorous immune response 26, 27. The Tg mice expressing the HLA class I gene were derived from homozygous C57BL/6 (H-2Kb) mice [22]. The HLA-DR1 transgene was inserted into B10.M/Sn (H-2f) mice,
In vitro proliferative response of human PBMC to TT and TT-derived peptides
Previous studies have defined TT-derived CD4+ TH epitopes that promiscuously bound to DR alleles as detected by in vitro primary immunization of human PBMC (Table 1). However, it was unclear which promiscuous TT epitope caused superior immune responses in the context of the broadest number of DR alleles. TT protein and five of the most promising TH epitopes were used to stimulate human T-cell proliferative responses in 43 randomly chosen donors (MHC haplotypes unknown). As a control, PBMC were
Discussion
Synthetic peptide-based CTL epitope vaccines have been developed as immunotherapeutics against a number of human diseases 13, 50, 51. In this report, the HLA-DR requirement for in vivo priming of HLA-A∗0201 restricted CTL responses has been examined. Initially, several TT immunodominant TH epitopes (TT639–652, TT830–843, and TT947–967) among five examined were shown to efficiently stimulate in vitro proliferation of PBMC as evaluated in 43 randomly chosen human donors. Ten additional HLA-typed
Acknowledgements
The authors would like to thank the following people for assistance in conducting the studies described in this report: Dr. Susan Kovats (Division of Immunology, COH) for help with PCR analysis and flow cytometry; Alice Huang for expert histochemical analysis of mouse tissues (Department of Anatomic Pathology, COH); Dr. Corinna La Rosa and Julie Papp, M.S. (Department of Hematology Research, COH) for providing fibroblasts, B-LCL, and titered CMV stocks; and Heather Adams, Donna Isbell, and Dr.
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This work is supported by grants from the Leukemia Society of America to D.J.D. (LSA #98-6116), and grants from the NCI (CA77544 and CA30206-Project III) and CA33572 as a Cancer Center core grant to the City of Hope.