The feasibility of a purified, inactivated dengue (DEN) vaccine made in Vero cells was explored. A DEN-2 virus candidate was chosen for production of a monotypic, purified, inactivated vaccine (PIV). Virus was harvested from roller bottle culture supernatants, concentrated, and purified on sucrose gradients. The purified virus was inactivated with 0.05% formalin at 22 degrees C. After inactivation, the virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody. Mice were completely protected against challenge with live, virulent virus after receiving two 0.15-microg doses of PIV. Monkeys vaccinated with three doses ranging as low as 0.25 microg demonstrated complete absence or a significant reduction in the number of days of viremia after challenge with homologous virus. These results warrant further testing and development of PIVs for other DEN virus serotypes.

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A panel of 11 murine monoclonal antibodies directed against dengue type 2 was evaluated for antigen specificity by dot immunobinding assay and Western blot analysis and for in vitro and in vivo biological activities. Nine of the 11 monoclonal antibodies reacted with viral E-glycoprotein based on the Western blot analysis; one reacted with a 36 Kd protein present in dengue-infected C6/36 mosquito cells. The nine E-glycoprotein-reactive monoclonal antibodies also neutralized dengue 2 virus in a plaque reduction assay. Of the neutralizing monoclonal antibodies, five passively protected mice in vivo against lethal intracerebral dengue 2 challenge. The protective monoclonal antibodies were directed against viral determinants that fell into at least three spatially separate families of epitopes on E-glycoprotein, the antigenicities of which were preserved after heat/detergent denaturation.

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