The dengue viruses are unique amongst flaviviruses in retaining a high proportion of the full length second envelope glycoprotein, prM. Despite evidence that the pr (cleaved precursor region) may generate antibody-dependent enhancement, which has been implicated in dengue viral pathogenesis, while M (membrane spanning region containing a truncated extra-envelope region) may generate protective immunity in animals, no epitopes on either region have been located.
The high sequence variability of dengue-2 virus prM suggests that by studying the biology and immunology of the polymorphisms, we may elucidate the mechanisms of viral virulence and protection.
Neutralization and virulence heterogeneity have been demonstrated within the main envelope (E) protein of some flaviviruses, but this has been poorly documented for the dengue viruses.
The envelope proteins (E & prM) of only single isolates of dengue-3 and dengue-4 viruses have been sequenced, despite these serotypes often being associated with severe disease.
We plan to sequence the genes encoding these proteins (E & prM) from strains of both serotypes that have shown clear differences in their pathogenic capacities.
Both linear and conformational epitopes on these proteins will be located using synthetic peptides and recombinant cDNA expression constructs. Amino-acid substitutions within the identified epitopes will be analysed to associate particular residues with either attenuation or virulence. This will be supported by immunizing mice with peptides containing these sequences and subsequently challenging with parent and heterologous virus strains to test their ability to either protect or enhance disease. Both synthetic peptides and recombinant proteins will be compared as diagnostic antigens and the polymerase chain reaction (PCR) will be as a method for the rapid identification of virulent dengue virus strains. This project will, therefore, define epitopes associated with protection or virulence on these viruses and act as a 'stepping stone' to the develpoment of a safe and inexpensive vaccine.
Funding SchemeCSC - Cost-sharing contracts