Identification of novel Plasmodium gallinaceum zygote- and ookinete-expressed proteins as targets for blocking malaria transmission

Abstract

The development of transmission-blocking vaccines is one approach to malaria control. To identify novel Plasmodium zygote- and ookinete-secreted proteins as targets of blocking malaria transmission, monoclonal antibodies (MAbs) were produced against parasite-secreted proteins found in Plasmodium gallinaceum ookinete culture supernatants. Four MAbs-1A6, 2A5, 2B5, and 4B6-were identified that bound to P. gallinaceum zygotes and ookinetes in diverse patterns in terms of spatial localization on parasites, time course of antigen expression, and Western immunoblot patterns. MAbs 2A5 and 4B6 recognized more than one protein band as detected by Western immunoblot of P. gallinaceum ookinete supernatants. Beginning at 0 h postfertilization, MAb 2A5 recognized a diverse set of antigens; at 10 h postfertilization, MAb 4B6 recognized several antigens as well. MAb 1A6 recognized a single approximately 17-kDa protein, and 2B5 recognized a single approximately 32-kDa protein at 15 h postfertilization. In membrane feeding assays to assess the effect of these MAbs on P. gallinaceum infectivity for Aedes aegypti mosquitoes, the addition of MAbs 1A6 and 2B5 to infectious blood meals significantly inhibited oocyst development in the mosquito midgut. In contrast, MAb 2A5 seemed to enhance infectivity. These results demonstrate that Plasmodium ookinetes secrete proteins (in addition to previously characterized chitinases) that may be targets for blocking malaria transmission. Future investigation of ookinete-secreted neutralization-sensitive molecules should provide valuable insight into mechanisms by which ookinetes exit the blood meal, penetrate and transverse the peritrophic matrix, and invade the mosquito midgut epithelium.

FIG. 1.

Immunofluorescence microscopy of P. gallinaceum ookinete cultures collected at 0, 10, 15, and 24 h postfertilization. Immunofluorescence was performed with MAbs 2A5 (A), 4B6 (B), 1A6 (C), 2B5 (D), and an antigen-irrelevant IgG isotype control (E). Bar, 5 μ m.

FIG. 2.

Western immunoblot analysis of P. gallinaceum ookinete culture supernatants collected 24 h postfertilization. Molecular masses are indicated in kilodaltons at the left.