CONSERVATION OF CRITICAL ED3 MONOCLONAL ANTIBODY BINDING RESIDUES AMONG DIFFERENT DENGUE 2 GENOTYPES

Trevor Pitcher^1,2, Michael Patterson², Gregory D. Gromowski^1,2, Alan Barrett, Ph.D. ^1,2

¹Department of Pathology ; ²Sealy Center for Vaccine Development, UTMB

Background: Dengue (DEN) is the most important mosquito borne viral disease in the world, causing upwards of 100 million infections per year. Previous studies have identified residues in DEN 2 envelope protein domain 3 (ED3) that are critical for type and complex specific antibody binding and high titer neutralizing (PRNT₅₀) antibodies. Methods: To demonstrate that critical monoclonal antibody (MAb) binding residues are conserved among different DEN 2 genotypes, four representative viruses were compared, representing different genotypes. Recombinant ED3 from each of these viruses was cloned into the expression vector pMal-c2x and expressed as a fusion protein with maltose binding protein. Substitutions of critical DEN2 type and DEN complex specific antibody binding residues, shown to dramatically affect antibody binding, were also introduced into each rED3. The rED3s were used in an ELISA assay to analyze the effects of these substitutions on type and complex specific MAb binding to the different DEN genotypes. These MAbs were also used to determine the PRNT₅₀ of each virus. Results: The physical Kd and PRNT₅₀ were shown to be different depending on the particular genotype. The binding of the type and complex specific MAbs to the various rED3s are currently being examined in ELISAs. Conclusions: Critical DEN2 type and DEN complex specific epitopes differ among the different genotypes. Supported by the NIH and John S Dunn Chair.