A vaccinia recombinant expressing dengue type 4 virus structural (C, M, E) and nonstructural (NS1, NS2a) proteins failed to induce a detectable immune response to the envelope (E) glycoprotein in cotton rats or rabbits. Because of the possibility that cell surface expression might increase immunogenicity, we employed a vaccinia virus vector to express the dengue E protein on the cell surface, using the RSV G glycoprotein as a carrier. Recombinant DNA constructs were prepared that encoded a truncated N-terminal segment of the RSV G glycoprotein fused to an 85% fragment of the (E) glycoprotein or to smaller E subfragments. These chimeric DNAs were inserted into vaccinia virus and the expression of E by recombinant vaccinia virus was analyzed. The expressed fusion protein containing 85% of E reacted with hyperimmune dengue virus antiserum by immunofluorescence assay, but was only found intracellularly. In contrast, cell surface expression of chimeric proteins containing a 34, 49 or 60% subfragment of E was demonstrated using antipeptide sera but these proteins did not react with hyperimmune dengue antiserum. Infection of cotton rabbits and rabbits with vaccinia recombinants expressing various chimeric proteins did not induce a detectable E antibody response. Mice immunized with the various vaccinia-chimeric E recombinants were partially resistant to lethal challenge with dengue type 4 virus, but protective efficacy varied markedly in two successive experiments, and a correlation was not observed between surface expression and protective efficacy. Surviving mice exhibited signs of encephalitis. In contrast, a vaccinia recombinant expressing: (i) the structural proteins (C, M, E) plus the first 2 non- structural proteins (NS1, NS2a), or (ii) only the 3 structural proteins, or (iii) only E induced complete resistance to lethal dengue virus challenge and most surviving mice did not develop signs of encephalitis. The latter observations indicate that expression of the dengue virus E and NS1 protective antigens in their mature on the cell surface is not required for induction of resistance.
