Regner, and M
Regner, and M. Our investigation also established the suitability of a novel flavivirus challenge model (2-microglobulin-knockout mice) for studies of the role of B-cell memory responses in vaccine protection. INTRODUCTION Japanese encephalitis (JE) computer virus (JEV) is usually a neurotropic flavivirus that can cause severe central nervous system (CNS) disease in humans and animals (examined in recommendations 1 and 2). It is a mosquito-borne pathogen that is prevalent in south and southeast Asia, China, and the Asia-Pacific region, where it is responsible for approximately 50,000 annual JE clinical presentations, with 20 to 30% resulting in death and 30 to 50% resulting in irreversible neurologic damage among survivors (3, 4). JE is Bupivacaine HCl usually primarily a disease of children since most adults in regions of endemicity show natural immunity, but it is also a health risk to travelers to regions of endemicity. Vaccination is the most important control measure against JE and has been highly successful in countries that have implemented national immunization programs since the availability of the first JE vaccine in the late 1960s. Nevertheless, vaccination has failed to halt the spread of JEV in Asia and the Asia-Pacific region (5), and transmission of JEV is likely to continue to increase in low-income countries (4). The first licensed JE vaccine was a mouse brain-derived formalin-inactivated antigen (JE-VAX) supplied from Japan for decades for internal and international use (examined in reference 6). In recent years, JE-VAX has been superseded by second-generation formalin-inactivated vaccines produced from cell culture-grown JEV or by live attenuated vaccines (examined in reference 7). However, JE-VAX remains the platinum standard for immunogenicity and security comparisons of new-generation vaccines against JE (8). Using JE-VAX as a comparator, we showed that JE-ADVAX, a Vero cell culture-grown inactivated JEV antigen (ccJE) (9), combined with Advax, a novel polysaccharide adjuvant derived from delta inulin (10), provided immunogenicity greatly superior to that of JE-VAX in mice and horses (11). In the same study, we also found that JE-ADVAX elicited levels of neutralizing antibody against serologically related flaviviruses of medical significance (West Nile and Murray Valley Bupivacaine HCl encephalitis viruses) that were indicative of cross-protective Bupivacaine HCl immunity, because they exceeded the titers against the homologous computer virus (JEV) generated by immunization with the platinum standard JE-VAX (11). The possible feasibility of cross-protective vaccination against multiple flaviviruses belonging to the JE serocomplex using a single antigen experienced previously been proposed only for live attenuated JE vaccines (12, 13) (examined in reference 14). In view of the excellent immunogenic properties of JE-ADVAX, it was of interest to delineate the immunological correlates underlying vaccine protection (examined in reference 15). In studies with knockout mice lacking B cells or CD8+ T cells or mice with poor persistence of neutralizing antibody or by passive transfer of immune effector cells from immunized donor to na?ve recipient mice, we show that JE-ADVAX mediates durable, protective immunity by induction of a long-lived memory B-cell populace that affords protection against JEV without the need for Bupivacaine HCl CD8+ T cells or pre-exposure neutralizing antibody. MATERIALS AND METHODS Viruses and cells. Vero (African green monkey kidney) cells were obtained from the American Type Culture Collection HDAC3 and were produced at 37C in a 5% CO2 atmosphere in Eagle’s minimal essential medium plus nonessential amino acids (MEM; Invitrogen) supplemented with 5% fetal bovine serum (FBS). Working stocks of JEV (strain Nakayama) were prepared as infected Vero cell culture supernatants (2 108 PFU/ml) and stored in single-use aliquots at ?80C. Computer virus titration was by plaque assay on Vero cell monolayers, as previously explained (16). Mice. C57BL/6 (B6), congenic B-cell-deficient (MT?/?) (17), and 2-microglobulin-deficient (2m?/?) (18) mice were bred under specific-pathogen-free conditions and supplied by the Animal Breeding Facility at the John Curtin School of Medical Research, The Australian National University or college (ANU), Canberra, Australia. Female mice were used in all experiments. All animal experiments were approved by and conducted in accordance with the ANU Animal Ethics Committee. Vaccines and adjuvant. Vaccines were formulated and administered to mice as previously explained (11). JE-ADVAX was a formulation of Vero cell culture-grown, inactivated.