Carboxy-terminally truncated dengue virus envelope glycoproteins expressed on the cell surface and secreted extracellularly exhibit increased immunogenicity in mice.

Recombinant vaccinia viruses expressing C-terminally truncated E's that ranged in length from 9 to 99% of the N-terminal sequence were constructed. The overall antigenicity of the E products was analyzed by radioimmunoprecipitation, using dengue virus hyperimmune mouse ascitic fluid (HMAF) or an anti-E peptide serum. Truncated E that was 79% or less in length did not bind HMAF efficiently, whereas E constructs greater than 79% were able to bind HMAF with high efficiency. The first 392 amino acids of the dengue type 4 virus E sequence, including the Arg-392 following the 79% E C terminus, appeared to be critical for proper antigenic structure required for efficient binding by HMAF. Truncated E's ranging from 59 to 81% in length were secreted extracellularly, whereas smaller or larger E's were retained intracellularly. Secreted E's contained carbohydrate side chains that were resistant to endoglycosidase H digestion, suggesting that the transport of E occurs via a pathway from the rough endoplasmic reticulum through the Golgi complex. 79% E-RKG (which possessed the three additional amino acids immediately downstream of 79% E) was expressed at a high concentration on the surface of recombinant virus-infected cells presumably being inserted into the plasma membrane by a hydrophobic C-terminal membrane anchor. Evaluation in mice of the protective efficacy of the various vaccinia virus E recombinants indicated that only truncated E's that were recognized efficiently by HMAF induced a high level of resistance to dengue virus encephalitis. 79% E-RKG which is expressed at a high concentration on the surface of infected cells was highly immunogenic when tested for induction of an E antibody response. This suggests that cell surface expression of 79% E-RKG was responsible for its enhanced immunogenicity. Finally, passive immunization studies indicated that serum antibodies to E played a major role in the complete or nearly complete resistance to dengue virus challenge induced by certain vaccinia virus-truncated E recombinants.