A possible explanation for the computer virus sequence-specific differences in the generation of epitope-specific responses may relate to differing pMHC avidity among different epitope variants. YF result in hemorrhagic fever (1C5). CGS 21680 HCl The envelope (E) and NS3 proteins are known to induce adaptive immune responses (1, 6C7). Flavivirus antigen-specific CD8+ T-cells are important in clearing computer virus from tissues and preventing computer virus persistence (8C11). Primary exposure to a computer virus primesCD8+ T-cells by virus-derived Rabbit Polyclonal to CENPA immunodominant peptides, presented in the context of MHC class-I by antigen presenting cells (APCs), lead to clonal differentiation and proliferation of effector cells followed by contraction and memory generation (12C15). Memory T-cells generated against subdominant epitopes of one computer virus may demonstrate plasticity in antigen-recognition upon secondary heterologous challenge leading to protective immunity or immunopathology (16C19). We previously exhibited that sequential contamination with different DENV serotypes induces sequence-specific growth of DENV cross-reactive CD8+ T-cells (20). Prime-boost immunizations using recombinant DNA vaccines or virus-like particles similarly enhance immune responses to inserted heterologous epitopes (21C24). The co-circulation and emergence of flaviviruses in many regions of the world point to the need for effective vaccination strategies against these viruses (1,2). Novel, live-attenuated chimeric flavivirus vaccines have been produced on a platform based on the nonstructural and core backbone of the YF 17D vaccine and the prM and E of heterologous flaviviruses, including JE, WN, and DENV. These computer virus vaccines have exhibited protection against homologous computer virus challenge in animal models and generation of adaptive B and T cell responses in human clinical trials (25C30). Neutralizing antibody responses to chimeric vaccine viruses have been shown to be specific to the computer virus from which the E gene is derived (31,32). CD8+ T cells have been found to be necessary for protection from primary dengue, WNV and JE computer virus infections in mouse models; WNV envelope-specific CD8+ T cells afford protection against lethal WNV challenge (8,9,11,33,34). The potential of chimeric vaccines to generate cross-reactive CD8+ T-cell responses to heterologous flaviviruses has not been studied. Here, we evaluated quantitatively and qualitatively the CD8+ T-cell responses to a subdominant flavivirus cross-reactive T-cell epitope around the E protein and to a conserved, immunodominant YF NS3 epitope, following primary or heterologous secondary contamination. We found that secondary immunization with heterologous chimeric flavivirus vaccines generated an enhanced cross-reactive CD8+ T-cell response that was dependent on the sequence of the infecting vaccine viruses. These results suggest that controlled exposure to multiple related flavivirus vaccines may lead to enhanced protection against related flaviviruses. MATERIALS AND METHODS Mice, viruses and immunization All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. 5C6 week aged female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were immunized CGS 21680 HCl intra-peritoneally (ip) with 1106 PFU ChimeriVax?-JE (YF/JE) or ChimeriVax?-WN (YF/WN) (from Acambis, Inc., now a part of Sanofi Pasteur), or YF (YF-Vax, from Connaught Laboratories, now a part of Sanofi Pasteur). Viruses were prepared and titered in Vero-81 cells (ATCC) (35). Kb and Db knockout mice (Taconic) were used to confirm MHC restriction of CD8+ T-cell epitopes. For primary immunizations, splenocytes were harvested at serial days post immunization (dpi) for acute responses and on day 28 for memory responses. To determine secondary CD8+ T-cell responses, 4C6 weeks following primary immunization, groups of 3 to 4 4 mice from each primary computer virus immunization were CGS 21680 HCl immunized with either homologous or heterologous viruses ip with 1106 PFU of computer virus. Peptides Crude peptides (20mer overlapping by 10 amino acids) spanning the envelope region of JE SA14-14-2 (GenBank Accession number “type”:”entrez-protein”,”attrs”:”text”:”AAK11279″,”term_id”:”12964701″,”term_text”:”AAK11279″AAK11279) and peptide truncations were synthesized by the University of Massachusetts Medical School Peptide Core Facility. WNE1 is usually a Db-restricted CD8+ T-cell epitope (11). Biology Workbench blast search revealed homologous sequences for other flaviviruses (Table 1). Variant E and YF NS3 peptides were synthesized at CGS 21680 HCl 90% purity by AnaSpec, Inc. Table 1 The heterologous envelope variants and conserved NS3 epitopes. thead th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Computer virus /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Sequence /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Homology /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Accession No. CGS 21680 HCl /th /thead JE SA14C14C2LGMGNRDFI100%”type”:”entrez-protein”,”attrs”:”text”:”AAK11279″,”term_id”:”12964701″,”term_text”:”AAK11279″AAK11279WNV NY 99LGM em S /em NRDF em L /em 78%”type”:”entrez-protein”,”attrs”:”text”:”AAW50577″,”term_id”:”57341359″,”term_text”:”AAW50577″AAW50577SLELG em TS /em NRDF em V /em 67.