ChimeriVax-WN02 is a novel live-attenuated West Nile virus (WNV) vaccine containing modified WNV premembrane (prM) and envelope (E) sequences inserted into the yellow fever 17D vaccine genome. established, highly immunogenic vaccines. West Nile virus (WNV), a mosquito-borne flavivirus, has gained increasing public health importance since its emergence in North America in 1999 [1, 2]. The spectrum of WNV disease ranges from asymptomatic infection to febrile illness to neuroinvasive disease that may lead to long-term sequelae or death [3, 4]. More than 11,000 cases of WNV neurologic disease with Diphenhydramine hcl > 1000 fatalities were reported in the United States in the past decade [1]. Even in the absence of central nervous system involvement, infection can cause substantial morbidity [5, 6]. The homology of WNV to yellow fever virus has been exploited in the development of a vaccine, ChimeriVax-WN02 (chimeric WN/YF), which comprises Diphenhydramine hcl WNV NY-99 premembrane (prM) and modified envelope (E) sequences inserted into the backbone of the yellow fever 17D (YF-17D) vaccine genome [7, 8]. Genetic variation in the WNV structural genes has increased since 1999, but most nucleotide changes in the envelope are transitions or silent mutations [9]. Phase 1 clinical trials of chimeric WN/YF demonstrated induction of neutralizing antibody as well as interferon (IFN-) production upon restimulation with WNV antigens [8]. Recent studies have highlighted the importance of CD8+ T cells in WNV infection. In mice, CD8+ T cells are necessary for protection against central nervous system disease [10C14]. In humans, patients with hematologic malignancies, those receiving antiCT cell therapies, and individuals homozygous for Rtp3 a mutation in the CCR5 chemokine receptor, which restricts leukocyte migration to the central nervous system, are at increased risk of disease and death following WNV infection [15C17]. In individuals vaccinated with chimeric WN/YF virus, we identified CD8+ T cell responses directed against a WNV envelope epitope that is also an immunodominant T cell target in naturally infected individuals [18C20]. We employed tetramer staining to measure the magnitude and resilience of CD8+ T cell responses to WNV envelope protein. We utilized phenotypic characteristics of memory phase T cells to examine the development of WNV envelope-specific T cells [21C24]. Finally, we assessed the functional durability of the response by assessing cytotoxic potential and cytokine production following rechallenge with WNV envelope antigen in vitro. MATERIALS AND METHODS Study Design The details of the Phase 1 study have been reported elsewhere [8]. Volunteers were immunized with chimeric WN/YF at 2 different doses (3 log10 plaque-forming units (PFUs) or 5 log10 PFUs), YF-17D vaccine, or inert placebo. Blood samples obtained on days 0, 14, 28, 90, 180, and 360 after immunization were separated and then cryopreserved until use. Study protocols were approved by the institutional review boards of the participating centers. Viruses Construction of chimeric WN/YF virus has been described elsewhere [7]. In Diphenhydramine hcl brief, the prM and E genes of the YF-17D vaccine strain were exchanged for those of the WNV NY-99 strain 385-99. Three attenuating mutations were introduced into the WNV E codon: E170 (leucine to phenylalanine), E336 (alanine to valine), Diphenhydramine hcl and E440 (lysine to arginine). Serum viremia was measured by plaque assay [8]. Construction of Recombinant Vaccinia Viruses The WNV E sequence was amplified from the full-length WNV complementary DNA [25] by use of GeneAmp XL polymerase chain reaction (PCR; Applied Biosystems) and cloned into the donor vector pDONR221 by use of a PCR cloning system with Gateway Technology (Invitrogen). The vaccinia transfer plasmid pMJ601 was modified to facilitate insertion of the WNV E gene (Gateway Vector conversion system; Invitrogen). Recombinant vaccinia viruses were generated by homologous recombination and propagated.