Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pf s25, a Leading Transmission-Blocking Vaccine Candidate

Abstract

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a Plasmodium falciparum multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP1₁₉) to P. falciparum MSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP1₁₉ Here, using the PfMSP1/8 construct, we further optimized the recombinant PfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate Pfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of Pfs25 block sexual-stage development in mosquitoes. The sequence encoding mature Pfs25 was codon harmonized for expression in Escherichia coli We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with the Pfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native Pfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of PfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

Keywords: MSP8 carrier protein; malaria; subunit vaccines; transmission blocking.

FIG 1

Targeted amino acid modifications within the PfMSP8 carrier domain of rPfMSP1/8 improve the yield of monomeric rPfMSP1/8(CΔS). (A) Schematic of the original pET28a-PfMSP1/8 construct (top) depicting two cysteine residues (red asterisks) within the PfMSP8 domain that were mutated to serine residues via site-directed mutagenesis, resulting in the construct pET28a-PfMSP1/8(CΔS) (bottom). (B) Lysates of E. coli transformed with pET28a-PfMSP1/8(CΔS), harvested before (T₀) or 3 h after (T₃) induction, were separated by SDS-PAGE (10% gel) under both reducing and nonreducing conditions, followed by Coomassie blue staining. (C) Immunoblot analysis of T₀ and T₃ lysates probed with rabbit anti-PfMSP1/8 IgG. (D and E) Purified rPfMSP1/8 (original) (lane 1) and rPfMSP1/8(CΔS) (modified) (lane 2) were separated by SDS-PAGE (10% gel) under nonreducing conditions, followed by Coomassie blue staining (3 μg/lane) (D) or immunoblot analysis (50 ng/lane) (E) of samples probed with rabbit anti-PfMSP1/8 IgG.

FIG 2

Modification of the PfMSP8 domain does not affect the immunogenicity of rPfMSP1/8(CΔS) relative to the original rPfMSP1/8 antigen. CB6F1/J mice (5/group) were immunized three times with 10 μg/dose of rPfMSP1/8 (black bars) or rPfMSP1/8(CΔS) (gray bars) formulated with Quil A (A) or Montanide plus CpG ODN (B) as an adjuvant. Sera collected following the final immunization were analyzed for antigen-specific IgG titers (means ± standard deviations) by an ELISA using plates coated with rPfMSP1/8, rPfMSP8, GST-PfMSP1₁₉(FVO), or GST-PfMSP1₁₉(3D7) antigens. ns, nonsignificant (P > 0.05).

FIG 3

PfMSP8 is expressed during P. falciparum gametocyte development but is absent on the surface of activated macrogametes. (A) Detection of PfMSP8 expression in fixed and permeabilized P. falciparum gametocytes (stages II to V) by an immunofluorescence assay with rabbit anti-rPfMSP8 IgG or control IgG followed by FITC-conjugated secondary antibodies. DAPI was used to stain parasite DNA. (B) Analysis of PfMSP8 expression on activated, live P. falciparum macrogametes by an immunofluorescence assay, as described above, with rabbit anti-rPfMSP8 IgG. Samples were costained with MAb 4B7, which is specific for Pfs25 (MAb 4B7), followed by TRITC-conjugated secondary IgG. DIC, differential interference contrast.

FIG 4

PfMSP8 is not a target of transmission-reducing antibodies. The functional activity of IgG purified from rabbit antisera raised against rPfMSP8 (n = 1) or rPfMSP1/8 (n = 3) was assessed in a P. falciparum standard membrane feeding assay at a concentration of 3.75 mg/ml. The graph depicts the average number of oocytes formed in the midgut of 20 mosquitoes per IgG sample. Positive and negative controls were MAb 4B7 and adjuvant control rabbit IgG, respectively.

FIG 5

Expression of chimeric rPfs25/8(CΔS) and unfused rPfs25. (A) Schematic depiction of expression constructs for the production of rPfs25/8(CΔS) and rPfs25. Expression plasmids for chimeric rPfs25/8(CΔS) or unfused rPfs25 were transformed into E. coli SHuffle T7 Express lysY cells. (B and C) rPfs25/8(CΔS) lysates harvested before (T₀) or 3 h after (T₃) induction were separated by SDS-PAGE (10% gel) under reducing conditions, followed by Coomassie blue staining (B) or immunoblot analysis (C) using rabbit-anti-rPfMSP8 IgG. The asterisk highlights rPfs25/8(CΔS) at the predicted size. (D and E) The expression of unfused rPfs25 was assessed as described above, on 12% polyacrylamide gels under reducing conditions, followed by Coomassie blue staining (D) or immunoblot analysis (E) using an anti-His MAb. The asterisk highlights rPfs25 at the predicted size.

FIG 6

Purification of the rPfs25-based vaccine. (A and B) Purified rPfs25 was analyzed by SDS-PAGE (12% gel) under both reducing (R) and nonreducing (NR) conditions, followed by Coomassie blue staining (3 μg/lane) (A) or immunoblot analysis (0.2 μg/lane) (B) using anti-Pfs25 MAb 4B7. (C to E) Purified rPfs25/8(CΔS) was analyzed by SDS-PAGE (10% gel) under both reducing and nonreducing conditions, followed by Coomassie blue staining (3 μg/lane) (C) or immunoblot analysis (0.4 μg/lane) using anti-Pfs25 MAb 4B7 (D) or rabbit anti-PfMSP8 (E).

FIG 7

Immunization with rPfs25 or rPfs25/8(CΔS) elicits high titers of Pfs25-specific antibodies. New Zealand White rabbits were immunized three times with rPfs25 (n = 4) (black bars), rPfs25/8(CΔS) (n = 4) (white bars), antigens formulated with Alhydrogel as an adjuvant, or the adjuvant alone. Sera collected 2 weeks following each immunization were analyzed for antigen-specific IgG titers (means ± standard deviations) by an ELISA using plates coated with rPfs25, rPfs25/8(CΔS), or rPfMSP8 antigens. The signal using adjuvant control sera was subtracted as the background. ns, nonsignificant (P > 0.05).

FIG 8

Immunization with rPfs25 or rPfs25/8(CΔS) elicits Pfs25-specific antibodies that recognize native Pfs25. (A) Recognition of native Pfs25 by pooled rabbit antisera generated against rPfs25, rPfs25/8(CΔS), or the adjuvant control was assessed by an immunofluorescence assay on activated, fixed macrogametes. Samples probed with MAb 4B7 were used as a positive control. Bound antibodies were detected with FITC-conjugated secondary IgG. (B) The purified macrogamete lysate was separated by SDS-PAGE (12% gels) under nonreducing conditions, followed by an immunoblot assay using pooled sera from rabbits immunized with rPfs25 (lane 1), rPfs25/8(CΔS) (lane 2), or the adjuvant alone (lane 3).