Recombinant P. falciparum sexual-stage antigens recreate native parasite epitopes. Pooled day 70 anti-sera (n = 5 BALB/c mice) generated to the panel of sexual-stage antigens were used to probe parasite epitopes by IFA. IFA identified unique staining patterns of fixed parasite epitopes by pooled anti-sera. P. falciparum NF54 strain stage V gametocytes (A) and gametes (B) were generated in vitro. NF54 strain ookinetes were dissected from infected A. stephensi mosquito midguts (C). Parasites were air-dried onto glass slides, fixed with 4% paraformaldehyde. For gametocyte staining (A) the cells were permeabilized with 0.01% Triton X-100 prior to immunostaining. All anti-sera were tested; only pooled anti-sera with detectable reactivity are shown. Stage-specific positive and negative controls were included with each IFA study to calibrate exposure settings; positive controls included Pfs48/54-specific monoclonal antibody 3E12 for the gametocyte and gamete stages and Pfs25-specific monoclonal antibody 4B7 for the gamete and ookinete stages. IFA was performed to evaluate serum recognition of gametocytes and gametes generated in vitro and ookinetes generated in vivo through mosquito blood feeding and dissection. Nine of 12 sera stained Triton X-100 permeabilized, intra-erythrocytic gametocyte stages (A). Five of 12 anti-sera stained non-permeabilized, exoerythrocytic gamete stages (B) and 7 of 12 anti-sera stained nonpermeabilized ookinetes (C). Anti-sera generated to hypothetical protein HYP3 failed to recognize any sexual lifecycle stage by IFA.

Nikolaeva D, Illingworth JJ, Miura K, Alanine DGW, Brian IJ, Li Y, Fyfe AJ, Da DF, Cohuet A, Long CA, Draper SJ, Biswas S. Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies. Mol Cell Proteomics. 2020 19(1):155-166.