Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission.

Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins

Immunoblot and Coomassie-blue stain of algae-produced Pfs25 and Pfs28 analyzed by SDS and Native-PAGE.

<p>Five micrograms of reduced and non-reduced affinity purified (A) a-Pfs25 and (B) a-Pfs28 were resolved by SDS-PAGE, transferred to nitrocellulose, and detected with anti-FLAG mAbs. (C) Five micrograms of reduced a-Pfs25, a-Pfs28, and BSA were resolved by SDS-PAGE and stained with Coomassie-blue. (D) Five micrograms of non-reduced a-Pfs25, a-Pfs28, and BSA were resolved by Native-PAGE and stained with Coomassie-blue. (R – reduced, N – non-reduced).</p

Immunoblot of reduced and non-reduced algae-produced Pfs25 and Pfs28 with monoclonal transmission blocking antibodies.

<p>Reduced and non-reduced a-Pfs25 and a-Pfs28 were resolved by SDS-PAGE, transferred to nitrocellulose, and detected with (A) anti-Pfs25 4B7 mAb and (B) anti-Pfs28-2D8 mAbs, respectively.</p

Analysis of antibodies from mice immunized with algae-produced Pfs25 or Pfs28.

<p>(A) ELISA titers of mouse anti-sera elicited by algae-produced Pfs25 and Pfs28. Mice were immunized with affinity purified a-Pfs25 or a-Pfs28 using complete Freund's adjuvant followed by boosters with incomplete Fruend's adjuvant by intraperitoneal injection. Pooled sera was serially diluted and tested in triplicate against the corresponding algae-produced Pfs antigen; error bars are one standard deviation. Prebleed sera were tested as a negative control; error bars are four standard deviations. (B–D) Western blot analysis of <i>P. falciparum</i> mixed sexual stage lysates with anti-Pfs25-4B7 mAbs, a-Pfs 25 antisera, or a-Pfs28 antisera. Reduced and non-reduced sexual stage lysates were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were probed with (B) anti-Pfs25-4B7 mAbs, (C) antibodies raised to a- Pfs25, and (D) antibodies raised to a-Pfs28. (R – reduced, N – non-reduced).</p

Indirect immunofluorescence using immune sera from mice immunized with algae-produced Pfs25 or Pfs28 on <i>in-vitro</i> cultured <i>P. falciparum</i> gametocytes, gametes, and zygotes.

<p>DNA was stained using DAPI (blue) and antibody binding was visualized using Alexa Fluor 488-conjugated rabbit anti-mouse IgG (green) for (A) a-Pfs25 antisera and (B) anti-Pfs25 4B7 mAbs. Scale bars, 1 µm.</p

Structural analysis of algae-produced Pfs25 and Pfs28.

<p>(A) Far-UV circular dichroism spectra of algae-produced Pfs25 (Gray) and Pfs28 (Black). Spectra shown are measured mean residue ellipticity (solid lines) and best fit (dotted lines) using CDSSTR. The contribution of each secondary structure to the overall spectra as predicted by CDSSTR is shown as fractions. (B) Analysis of protected cysteines in a-Pfs25 by tandem mass spectrometry. The peptide sequence of Pfs25 from Ala₂₂ to Thr₁₉₃ is shown with EGF-like domain 1 (blue), 2(pink), 3(green), 4(orange). Peptides detected by mass spectrometry are underlined and protected cysteines are marked by stars. Predicted disulfide linkages between cysteines are marked numerically (i.e. the two cysteines labeled 1 form a dilsulfide linkage and so on). The epitope recognized by anti-Pfs25 4B7 mAb is indicated.</p