A plant-produced Pfs230 vaccine candidate blocks transmission of Plasmodium falciparum

Abstract

Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine.

Figures

Fig. 1.

Pfs230 genetic organization and construct design. (A) Schematic representation of predicted structural motif for Pfs230, including the approximate amino acid range of each relevant region or domain. The cleaved portion of Pfs230 contains the 25 glutamic acid repeat region, E, and the 16 tandem EEVG repeat region and precedes the seven paired cysteine motif domains, CM1 to CM7. (B) 230CMB includes aa 444 to 730 and contains the PR-1a signal peptide at the N terminus and a 6-His tag and the KDEL ER retention signal at the C terminus (light gray boxes). (C) Diagram of the launch vector for target expression containing the Agrobacterium left border (LB) and right border (RB) sequences, the plant promoter (Prm) and terminator (Trm) regulatory elements, the viral vector launch sequences, and the Pfs230CMB target sequence.

Fig. 2.

Expression analysis and in vitro characterization of the purified 230CMB antigen produced for immunological evaluation. (A) Crude plant extract consisting of total soluble protein with 0.5% Triton X-100 (TSPT) and total protein (TP) were resolved by SDS-PAGE, transferred to PVDF membrane, detected with anti-4-His MAb, and compared to different amounts of the standard (plant-produced hemagglutinin from the A/Indonesia/05/05 strain of influenza virus [HAI]). (B) Increasing amounts of purified 230CMB were resolved by SDS-PAGE and stained with Gel Code Blue. Size comparisons to MagicMark XP (A) or BenchMark (B) molecular size markers were made. The values to the left of panels A and B are molecular sizes in kilodaltons. (C) SEC of 230CMB followed by UV and light scattering detection. The solid black trace is UV280, the thin dotted trace is the light scatter signal (Rayleigh ratio), and the markers indicate the calculated molar mass over the given elution volume.

Fig. 3.

IgG responses elicited by the 230CMB vaccine in rabbits using either complete Freund's adjuvant (CFA) followed by incomplete Freund's adjuvant (IFA) subcutaneously (SC) or Alhydrogel intramuscularly (IM). Serum 230CMB-specific IgG titers were measured by ELISA using samples collected before (Preimmune) antigen administration, and on days 28, 42, and 56 postvaccination. Reciprocal serum dilutions that gave a mean OD value greater than 4-fold over the background were determined as the endpoint titers. Data are shown as mean serum IgG endpoint titers ± standard deviations.