TARGETING PLASMODIUM INVASION PATHWAYS IN MOSQUITOES TO BLOCK MALARIA TRANSMISSION
Abstract
Malaria remains a devastating disease. Transmission-blocking vaccines (TBVs) are being considered as a promising approach to eliminate Plasmodium infection. However, the challenges in developing such a vaccine are paramount, since the clinically relevant species of Plasmodium are transmitted by a number of different Anopheles mosquito vectors. Through association studies, we discovered FREP1, a mosquito midgut protein that facilitates P. falciparum parasite transmission. Our biochemical characterization of FREP1 discovered that this protein is secreted as a tetramer, expressed by midgut cells and located in the peritrophic matrix (PM) of mosquito midgut. Molecular analysis revealed that FREP1 anchors parasites to the mosquito PM, assisting ookinetes as a midgut receptor for Plasmodium migration from the blood bolus and subsequent invasion of the midgut epithelium. Since FREP1 is readily accessible to antibodies co-ingested with blood, it is a suitable antigen for targeting by TBV. Sequence comparison of orthologs showed that the fibrinogen-like (FBG) domain of FREP1 is highly conserved (>90% identical) among Anopheles species from different continents, suggesting that anti-FBG antibodies may block malaria transmission to all anopheline mosquitoes. Using standard membrane-feeding assays (SMFA), we showed that anti-FREP1 polyclonal antibodies significantly blocked transmission of P. berghei and P. vivax to An. gambiae and An. dirus respectively. Furthermore, in vivo studies of mice immunized with purified FBG showed that our experimental TBV effectively blocks P. berghei transmission to An. gambiae (>75%), without triggering immunopathology or inducing responses against mouse or human fibrinogens. Anti-FBG serum from the immunized mice also reduces P. falciparum infection of An. gambiae mosquitoes by more than 81% during SMFA, meeting TBV criteria for clinical trials. Finally, I showed that the FBG domain directly interacts with Plasmodium gametocytes and ookinetes, revealing the molecular mechanisms of the transmission-blocking activity of anti-FBG antibodies. FBG also binds to peritrophic matrix, and the N-terminal region of FREP1 keeps FREP1 as tetramers. Collectively, our data support that FREP1-mediated Plasmodium transmission to mosquitoes is a conserved pathway, and that the targeting of the FBG domain of FREP1 will limit the transmission of multiple Plasmodium species to multiple Anopheles species. In summary, I reported here the establishment of a high-level secretion system using mosquito FREP1 signal peptide to secrete recombinant heterologous proteins. I have elucidated FREP1 molecular mechanisms as an ookinete midgut receptor that facilitates parasite invasion of the mosquito midgut, and I have determined that the highly conserved functional FBG domain of FREP1 is a broad-spectrum transmission blocking vaccine antigen.
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