Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

Abstract

Recombinant Plasmodium falciparum merozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins in Escherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

Plasmodium falciparum Merozoite Surface Protein 3

“…While preparing recombinant MSP3F and MSP3N for immunological characterization studies, we observed that these proteins formed oligomers of high molecular masses (20). The presence of large molecular mass species of MSP3 has been described previously and attributed to the presence of a leucine zipper-like sequence in the C terminus of the protein (7).…”

“…These plasmids were transformed in BL21 (DE3) and expressed as soluble proteins. These recombinant proteins were purified under similar conditions as described previously (20). The purified proteins were dialyzed in 20 mM Tris, 150 mM NaCl, pH 8.0, and stored at Ϫ80°C for further experiments.…”

“…Thin smears of the above isolated merozoites were fixed with chilled methanol for confocal microscopy and imaged under confocal microscopy as described earlier (26). Slides were co-immunostained with MSP3 polyclonal sera (20) at dilutions 1:1000 along MSP3 monoclonal Abs, Rh2b (1:100) as rhoptry marker, EBA-175 (1:100) and AMA-1 (1:500) as microneme marker, MSP5 (1:500) as merozoite surface marker, in blocking buffer for 1 h (27). For another set of experiments, freshly isolated merozoites were treated with cytochalasin D followed by addition of pre-warmed fresh erythrocytes.…”

“…The full-length P. falciparum MSP3 (MSP3F) and a large N-terminal fragment MSP3(21-238) (MSP3N) from the 3D7 strain of P. falciparum were expressed in Escherichia coli and purified by immobilized metal affinity chromatography, followed by ion exchange chromatography, as described previously (20). The C-terminal hexahistidine-tagged polypeptides MSP3(191-312) (MSP3C) , MSP3(219 -312) (MSP3Cb), and MSP3(288 -354) (MSP3C) were amplified from the MSP3F plasmid using NcoI and XhoI restriction sites and cloned in pET28a vector.…”

The origin and diversification of the merozoite surface protein 3 (msp3) multi-gene family in Plasmodium vivax and related parasites

“…Domain architecture for the P. falciparum msp3 family has been explored previously (Gondeau et al, 2009; Imam et al, 2011). Although one P. falciparum MSP3 protein (PfMSP3.1) shares a region of heptad repeats resulting in predicted coiled-coil tertiary motifs with P. vivax MSP3, the other PfMSP3 proteins lacked the coiled-coil motifs (Jiang et al, 2013).…”