Immunity & infection
Antibodies have been implicated in both protection and pathology of dengue virus infections. However, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. To precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level.
This method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy- and light- variable regions into expression vectors containing the defined constant region followed by transient co-transfection of HEK 293A cells with the heavy and light chain expression vectors made from genes arising from the same cell.
Thus far, using this powerful technology, for the first time in India, we have made 73 number of human monoclonals, of which 36 are specific to dengue and 12 neutralize dengue and zika virus at various concentrations. All the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope.
Together, with the ongoing comprehensive analysis of the B cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications.