N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity

Abstract

The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.

Figures

Fig. 1.

Pfs230 primary structure and design of constructs. (A) Schematic representation of predicted structural motifs for Pfs230. SP represents a signal peptide. The cleavage site at amino acid (aa) 443 represents the site at which processing of Pfs230 occurs during gamete formation. The region comprising amino acids 443 to 588 refers to the pro domain. CM1 through CM7 (regions shaded in gray) represent CM domains as described by Williamson et al. (43). Amino acid positions (arrows) 589, 918, 1285, and 3135 represent the starts of CM1, CM2, and CM3 and the end of CM7, respectively. Also, domains I through XIV, as described by Gerloff et al. (11), are in parentheses. (B) Construct Pfs230C spans the pro domain through domain III and comprises amino acids 443 to 1132. Construct Pfs230C2 spans the pro domain through domain II and comprises amino acids 443 to 915. Construct Pfs230C1 spans the pro domain and domain I and comprises amino acids 443 to 715. Construct Pfs230C0 spans the pro domain only and comprises amino acids 443 to 588.

Fig. 2.

SDS-PAGE analysis of the purified truncated recombinant Pfs230 (rPfs230) proteins expressed. The different truncated Pfs230 proteins, Pfs230C0 (C0), Pfs230C1 (C1), Pfs230C2 (C2), and Pfs230C (C), were expressed in the wheat germ cell-free system and separated on an SDS-12.5% polyacrylamide gel under reducing condition and stained with Coomassie brilliant blue (arrows indicate the expected truncated Pfs230 proteins). The extra bands other than those indicated by arrows in lanes C2 and C are translation products due to premature termination of translation.

Fig. 3.

IgG responses elicited by immunization with different truncated forms of Pfs230 in rabbits. (A). Serum IgG titers in samples collected before antigen administration (pre) and on day 56 postimmunization using recombinant Pfs230C0, -C1, -C2, and -C as ELISA capture antigens. (B). Serum IgG titers in samples collected on day 56 postimmunization using native, parasite-derived Pfs230 as the plate antigen. Reciprocal serum dilutions that gave a mean absorbance at 415 nm of 0.5 were determined as the endpoint titers. OD, optical density.

Fig. 4.

Western blot analysis using antisera against different truncated forms of Pfs230, Pfs230C0 (C0), Pfs230C1 (C1), Pfs230C2 (C2), and Pfs230C (C). Extracts prepared from stage V gametocytes of the P. falciparum NF54 line were separated on SDS-12.5% polyacrylamide gels under nonreducing condition and transferred onto PVDF membrane. Proteins on PVDF membranes were immunostained with either rabbit anti-Pfs230C0, -C1, -C2, and -C sera (lanes C0, C1, C2, and C in the left panel) or the corresponding preimmune (Pre) sera (right panel) (arrow). The relative molecular masses of the proteins were estimated with reference to Precision Plus Protein Standards (Bio-Rad, Hercules, CA). To ensure that equal amounts of the protein samples were loaded into the lanes for Western blot analysis, the membranes were simultaneously probed with anti-PfHSP70 mouse MAb (4C9) as a quantitative marker of parasite protein (35) (arrowhead).

Fig. 5.

Reactivity of antisera against Pfs230C0, -C1, and -C2 and Pfs230C in immunofluorescence microscopy. Samples prepared from stage V gametocytes (upper panels) and gametes (lower panels) of the P. falciparum NF54 line were immunostained with the antisera indicated above the panels. Immunostained images were visualized with Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). Scale bars, 5 μm.