Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230

Abstract

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria.

Keywords: Pfs230; Pichia pastoris; malaria; protein structure; recombinant protein expression; transmission blocking; vaccine; yeast.

FIGURE 1.

Production of recombinant Pfs230 proteins. A, schematic of native Pfs230 based on a report by Gerloff et al. (24) and recombinant Pfs230D1–2, Pfs230D1H, and Pfs230D1M, as well as, for B-H, the biochemical and biophysical characterizations of the different recombinant forms. B, Coomassie Blue-stained SDS-PAGE analysis of recombinant forms of Pfs230. C, analysis of the Pfs230D1M intact mass by LC/MS. D, comparative analysis of the ellipticity of Pfs230D1M and Pfs230D1H by far-UV circular dichroism. E and F, analysis of Pfs230D1M by SEC-MALS-HPLC and RP-HPLC, respectively. G and H, analysis of Pfs230D1–2 by SEC-MALS-HPLC and RP-HPLC, respectively. I, Western blotting analysis with Pfs230-specific mAb 4F12 against native gametocyte lysate (left panel) or recombinant forms of Pfs230 (right panel) under both non-reduced and reducing conditions, respectively. Notations in panel A include Roman numerals showing 14 distinct cysteine domains, alpha numeric numbers represent the number of cysteines in each domain, and the gold-gray color represents a double domain as defined by Gerloff et al. (24). Asterisks denote position of cysteines within domains 1 and 2. Boundary for the epitope recognized by the Pfs230D1-specific mAb 4F12 is shown in panel A.

FIGURE 2.

Circular dichroism analysis of Pfs230D1M in aqueous solution. The ellipticity (deg cm²/dmol) was plotted as a function of wavelength (nm). Raw data measured in millidegrees was converted into ellipticity (deg cm²/dmol). Spectra were obtained at increments from 20 to 80 °C and then at 20 °C following cooling for protein renaturation.

FIGURE 3.

Surface reactivity of antibodies against Pfs230 and Pfs25 on live gametes. A, modified surface immunofluorescence assay analysis using Pfs230- and Pfs25-specific mAbs 4F12 and 4B7, and B, rabbit Pfs230D1M antiserum alone or in combination with Pfs25-specific mAb 4B7. C, live surface immunofluorescence assay analysis is shown for rabbit Pfs230D1M antiserum. Scale bar is equal to 2 μm.

FIGURE 4.

Ribbon diagram and amino acid substitutions corresponding to minor allelic variants identified in Pfs230D1. A, ribbon diagram of a Pfs230D1M comparative model. The cysteines participating in the two predicted disulfide bonds and the amino and carboxyl termini of the Pfs230D1M construct are labeled. B, the minor allelic variants are identified on a surface plot of Pfs230D1M: G605S, green; K661N, orange; and others, magenta.