The nonstructural protein 1 (NS1) of dengue virus has been shown by others to induce protective immunity in mice. The use of NS1 for immunization against infection has one theoretical advantage over immunization with the other dengue protective antigen, the envelope glycoprotein (E). It is possible that antibodies against the envelope protein might cause immune enhancement of disease during subsequent infection by a heterotypic dengue virus. For this reason we initiated a study of the immunogenicity of dengue type 4 NS1. A series of overlapping oligopeptides that span the entire NS1 amino acid sequence were synthesized. In addition, we constructed recombinant vaccinia viruses for expression of: (i) the entire NS1, alone or in combination with other dengue products, or (ii) subfragments of NS1. Analysis of the binding of NS1 specific antibodies to the peptides by ELISA and immunoprecipitation of subfragments of NS1 indicated that the amino terminal third of the NS1 protein contains the major antigenic epitopes. Studies of the expression of NS1 by another series of vaccinia recombinants demonstrated that the hydrophobic sequence preceding NS1 is a functional signal whether it is located at the amino terminus or at internal position of an expressed polyprotein. In addition, the downstream NS2a sequence was found to be required in cis for the normal cleavage of the NS1/NS2a junction.
