Human infections with Plasmodium spp. lead to around 500,000 deaths annually. With the high fatality rate and increasing resistance to available therapeutics an effective vaccine is needed. One of several possible vaccine strategies targets sporozoites, the immature parasite transmitted from mosquitoes to humans. Antibodies targeting sporozoites through an amino acid repeat region, (NANP)n, found within a sporozoite surface protein, have been proposed as capable of conferring protection against infection. Yet relatively little is known of this important immune response.
To begin to dissect this response we have taken a protective IgG antibody, 2A10, which targets the (NANP)n repeat and tested whether binding is dependent on somatic hypermutation and class switching. We created and compared recombinant IgM and IgG antibodies with either the germline or 2A10 fragment antigen binding region (Fab). By ELISA analysis there was no difference in ability to bind (NANP)n antigen between recombinant germline and 2A10 IgM antibodies, whereas recombinant 2A10 IgG showed greater binding with antigen than germline IgG. These results suggest that binding is improved through somatic hypermutation, and that the pentameric structure of IgM may confer increased avidity to germline Fab thereby compensating for decreased affinity.
To investigate the development of B cells responsible for generating protective antibodies we have developed tetramers capable of identifying (NANP)n-specific B cells. With this tetramer tool alone we have identified NANP specific B-cells from spleens and bone marrow of (NANP)n vaccinated mice. When combined with magnetic bead enrichment we are able to detect and characterize these antigen specific cells at a frequency of ~1 in 106. We anticipate that this tool will enable us to detail comprehensively B-cell responses in humans to both natural infection and vaccination, and delineate the features of a protective immune response.