EPITOPE MAPPING OF ENVELOPE PROTEIN OF DENGUE 2 VIRUS

Abstract

This project aimed to identify neutralising epitopes on dengue 2 virus by isolating neutralisation escape mutant viruses and sequencing the genome coding .for the envelope (Ii) protein of neutralisation escape mutants resistant to neutralising E protein specific mouse monoclonal antibodies. Phenotypic changes associated with the ability of these viruses to escape neutralisation by mouse monoclonal antibodies were also investigated.

Neutralisation escape mutant viruses were selected by passaging dengue 2 virus in baby hamster kidney (BHK) cells in the presence of one of the following mouse monoclonal antibodies (8C6, 5Hl2, 4E6, 6B9, 6B2 or 10F2). Virus was also passaged in the absence of selecting antibodies to determine the effect of cell passage alone. Cell passage alone induced changes in all passage control viruses which reduced the ability of the monoclonal antibodies to neutralise viral infection of BHK cells. In only two instances (with antibodies 6B2 and 10F2) were antibodies able to cause significant neutralisation of dengue 2 virus passaged in BHK cells in the absence of antibody.

Changes occurred at amino acid 311 (E to G) in virus which escaped from neutralisation by antibody 6B2 and at amino acid 69 (T to I) in virus which escaped from neutralisation by antibody I0F2. Although these two antibodies still continued to bind to their corresponding neutralisation escape mutant virus, they did so with a lower affinity when bound to wild type virus. This suggested that the amino acid substitutions occurred at the outer edge of the antibody "footprint".

Both antibodies (6B2 and 10F2) inhibited attachment of wild type and passage control virus to BI-IK cells only slightly, suggesting that they were not binding to the receptor binding determinant and neutralisation may have been occurring at a stage in the virus infection cycle after attachment of virus to its cell surface receptor.

MAb 6B2 was not able to inhibit fusion of C6/36 cells by wild type dengue 2 virus or the passage control (6B2N). In addition, 6B2M virus, in the absence of antibody, had a similar fusion pattern as its corresponding passage control virus. On the other hand, MAb 10F2 was able to inhibit fusion of C6/36 cells infected with dengue 2 passage control virus and the mutant viruses selected with this antibody were unable to fuse C6/36 cells. This suggested a role for the region of the E protein surrounding amino acid 69 in the fusion process.

The amino acid substitution (T to I) at amino acid 69 also rendered dengue 2 virus more sensitive to elevated temperatures a wild type, passage control or mutant viruses with an amino acid substitution (E to G) at amino acid 311. This indicated that the amino acid change at position 69 might cause structural instability. The localisation of epitopes involved in neutralisation of dengue 2 virus and the identification of properties of these regions will aid future development of safe, stable and effective dengue vaccines.