The focus of this proposal is to develop strategies that permit the rapid and efficient transfer of HCMV specific immunity from a bone marrow to an immunosuppressed recipient resulting in protective immune responses that limit or prevent disease. This approach is based on the development of subunit vaccines containing immunogenic viral proteins or peptides. The clear motivation for developing a simple and effective subunit viral protein vaccine is to provide an acceptable alternative to traditional pharmacologic based anti-viral therapy. Our approach will also provide a potentially simpler and cheaper alternative to the approach of adoptive transfer of ex vivo expanded CMV-specific CD8+ CTL from the bone marrow donor into the recipient. Since the HCMV matrix protein pp65 has been previously shown to be the target of a CD8+ CTL response in a large percentage of seropositive individuals, it will be the focus of strategies to define conditions of immunization which result in a widely applicable vaccine preparation. We will utilize the recently developed strategy of defining a minimal cytotoxic epitope (MCE) of a protein, as a means of priming a memory CTL response to the virus that encodes the protein in vitro using peripheral blood of CMV seropositive individuals. Narrowing our search of cytotoxic epitopes specific to several predominant HLAA alleles form HCMV pp65 will permit an early test of the effectiveness of the peptide vaccine strategy. We will determine the ability of an MCE from pp65 to activate memory CTL in a murine model of HCMV recognition. Alternatively, we will also explore whether previously characterized immunologic adjuvants are capable of introducing pp65 full length protein into the Class I protein degradation pathway, thereby causing the development of a primary CD8+ CTL response to the protein in mice. The strength and duration of the CTL response to HCMV pp65 in adjuvant+protein immunized mice will be measured after multiple inoculations of the immunostimulating complex. An adoptive transfer model of CTL immunity to HCMV pp65 will be developed that will combine our two proposed strategies of immunization; adjuvant+protein in the donor, and HLA restricted MCE in the immunosuppressed recipient. HLA-A2.1/Kb transgenic mice will be multiply immunized with adjuvant+protein(pp65), and bone marrow from them will be adoptively transferred into irradiated syngeneic recipients. The recipient's immune response to pp65 will be measured with and without a follow-up peptide immunization. This protocol will closely resemble actual conditions after BMT, and provide a realistic model of the ability of the immunization strategies posed here to provide durable immunity to HCMV in the critical time period after transplant for the immunosuppressed patient. These studies have broad implications for modulation of BMT recipient immunity by manipulation of the donor prior to transplantation, as well as for the general utility of protein vaccines in other groups of patients susceptible to severe HCMV-related disease.
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