Antigen discovery has focused on using human serum samples collected from individuals that are naturally exposed to malaria, and who appear to develop effective immunity that prevents gametocytemia, or that blocks parasite transmission to mosquitoes, as tools to identify candidate vaccine antigens. Specifically, serum samples that have activity of interest, are compared to sera that lack this activity, for their ability to select or recognize individual recombinant proteins constructs in P. falciparum expression libraries. Recombinant proteins identified through differential screening are then prepared as immunogens and tested for their ability to induce effective anti-gametocyte or transmission blocking antibodies. 1. Phage Display Subtractive Screening was performed using field sera having no transmission blocking activity for negative panning followed by positive panning with four different sera having natural transmission blocking activity individually. Using this approach sixteen different TBV candidates were selected which were recognized by at least two different sera having natural transmission blocking activity. Of this the top three TBV candidates were recognized by all four positive sera. 2. Genes corresponding to the top nine candidates were synthesized and cloned into mammalian expression vector for DNA immunization of mice to examine their transmission blocking activity by standard membrane feeding assay (SMFA). 3. Sera and purified IgG from immunized mice for the top candidate showed transmission blocking activity comparable to the positive control Pfs25. 4. To examine these candidates further the top three candidates recognized by all four positive sera was expressed in E.coli. 5. We are in collaboration with NMRC to generate phage nanoparticle based vaccines expressing the current TBV antigens Pfs25 and Pfs230 on the surface. 6. The E.coli expressed and purified proteins are currently being used to immunize mice either individually or in combination with either Pfs25 or Pfs230. The results from these studies will confirm the transmission blocking activity of the top candidate as well as its ability to add to the activity shown by either Pfs25 or Pfs230.
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