A transmission-blocking vaccine (TBV) against Plasmodium falciparum is likely to be a valuable tool in a malaria eradication program. Pfs230 is one of the major TBV candidates， and multiple Pfs230-based vaccines induced antibodies， which prevented oocyst formation in mosquitoes as determined by a standard membrane-feeding assay (SMFA). Pfs230 is a >300 kDa protein consisting of 14 cysteine motif (CM) domains， and the size and cysteine-rich nature of the molecule have hampered its production as an intact protein. Except for one early study with maltose-binding protein fusion Pfs230 constructs expressed in Esherichia coli， all other studies have focused on only the first four CM domains in the Pfs230 mo... More
A transmission-blocking vaccine (TBV) against Plasmodium falciparum is likely to be a valuable tool in a malaria eradication program. Pfs230 is one of the major TBV candidates， and multiple Pfs230-based vaccines induced antibodies， which prevented oocyst formation in mosquitoes as determined by a standard membrane-feeding assay (SMFA). Pfs230 is a >300 kDa protein consisting of 14 cysteine motif (CM) domains， and the size and cysteine-rich nature of the molecule have hampered its production as an intact protein. Except for one early study with maltose-binding protein fusion Pfs230 constructs expressed in Esherichia coli， all other studies have focused on only the first four CM domains in the Pfs230 molecule. To identify all possible TBV candidate domains， we systematically produced either single-CM-domain (a total of 14)， 2-CM-domain (7)， or 4-CM-domain (6) recombinant protein fragments using a eukaryotic wheat germ cell-free expression system (WGCFS). In addition， two more constructs which covered previously published regions， and an N-terminal prodomain construct spanning the natural cleavage site of Pfs230 were produced. Antisera against each fragment were generated in mice and we evaluated the reactivity to native Pfs230 protein by Western blots and immunofluorescence assay (IFA)， and functionality by SMFA. All 30 WGCFS-produced Pfs230 constructs were immunogenic in mice. Approximately half of the mouse antibodies specifically recognized native Pfs230 by Western blots with variable band intensities. Among them， seven antibodies showed higher reactivities against native Pfs230 determined by IFA. Interestingly， antibodies against all protein fragments containing CM domain 1 displayed strong inhibitions in SMFA， while antibodies generated using constructs without CM domain 1 showed no inhibition. The results strongly support the concept that future Pfs230-based vaccine development should focus on the Pfs230 CM domain 1.
