Chimeric dengue type 2/type 1 viruses induce immune responses in cynomolgus monkeys. Southeast Asian

Abstract. Chimeric dengue type 2/type 1 (DEN2/1) viruses, which contain the structural genes of the dengue-1 (16007) parental virus and the nonstructural genes of the DEN2-PDK53 virus, have been constructed. These DEN2/1 viruses induce high levels of DEN1 virus-specific neutralizing antibodies in mice. In this study, the DEN2/1 viruses induced DEN1 virus-specific neutralizing antibodies without the development of viremia in cynomolgus monkeys. Dengue virusspecific IgM antibodies were detected in the sera of the immunized animals as early as 3 days post-immunization. After challenge with the DEN1-16007 wild-type virus, only a low level of viremia was detected in chimeric DEN2/1 virus-immunized monkeys. A second challenge, with DEN2-16681 virus, was given while the levels of DEN2-specific neutralizing antibodies were very low: infectious Dengue 2 virus could not be detected in sera of the monkeys. A correlation between the level of neutralizing antibody and the incidence of viremia could not be found. In addition, there was no significant increase in the levels of interferon gamma and soluble interleukin 2 receptor in the sera of the challenged monkeys, which suggests a reduction in immunopathogenesis caused by T-cell activation. Our findings suggest that DEN2/1 viruses may used as a liveattenuated candidate vaccine because of their safety, broad immunogenicity, and lower immunopathogenicity

Dengue 2 PDK-53 Virus as a Chimeric Carrier for Tetravalent Dengue Vaccine Development

Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK(2) cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines

Dengue virus-specific memory T cell responses in human volunteers receiving a live attenuated dengue virus type 2 candidate vaccine

A live attenuated dengue virus type 2 candidate vaccine (16681-PDK53) was evaluated in a phase I trial in 10 nonimmune adult volunteers. The dengue virus-specific memory T cell responses were analyzed as part of this study. Dengue virus-specific T cell proliferative responses were observed in all subjects after stimulating their peripheral blood mononuclear cells with live viruses or noninfectious viral antigens. The highest proliferative response was against dengue virus type 2, although cross-reactivity with other flaviviruses was detected to a lesser degree in some subjects. Dengue virus type 2-specific CD4+ and CD8+ cytotoxic T lymphocytes were generated in all vaccinees. This study investigated whether the candidate vaccine was efficacious in inducing dengue virus-specific CD4+ and CD8+ T cell memory after a single immunization in nonimmune recipients