Malaria remains one of the most widespread parasitic diseases in the world with more than 40 per cent of humans under risk of contracting this life-threatening disease. To eliminate malaria, tools are needed that interrupt Plasmodium human-to-mosquito transmission. The 6-cysteine protein family is conserved across Plasmodium species. Members of this family of proteins are among the most abundant surface antigens and play critical roles in parasite transmission, evasion of the host immune response and host cell invasion. The 6-cysteine protein Pfs230 is present on the surface of sexual-stage parasites, involved in fertilization of male and female gametes, and is a leading transmission blocking vaccine candidate. Here we generated and characterized a panel of nanobodies that bind Pfs230 with high affinity and map into two separate epitope groups. To demonstrate functionality in the biological context, we use our Pfs230-specific nanobodies to detect Pfs230 from parasite material via western blotting and immunofluorescence microscopy. Nanobodies of both epitope groups reduce oocyst formation in the mosquito midgut showing potent transmission blocking activity. Using X-ray crystallography, we determined two crystal structures of Pfs230 in complex with our transmission blocking nanobodies at 1.7 and 2.1 Å resolution. The crystal structures suggest that nanobody binding to Pfs230 may have an effect on the binding of the Pfs230 prodomain and potentially provide a structural mechanism for inhibition of Pfs230 during parasite transmission.
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