Purification, Characterization, and Immunogenicity of a Disulfide Cross-Linked Plasmodium vivax Vaccine Candidate Antigen, Merozoite Surface Protein 1, Expressed in Escherichia coli

Abstract

The Plasmodium vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami(DE3), of Escherichia coli with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional E. coli BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to >99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against P. vivax MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.Plasmodium vivax is one of the two major human malaria parasites and alone is responsible for 40 to 50% of all malaria cases in Latin America and southeastern Asia. The emergence of drug-resistant P. vivax strains (1) has emphasized the need for a vaccine. Progress toward a vaccine to prevent P. vivax infection is severely constrained by the availability of recombinant P. vivax antigens suitable for efficacy trials in humans. The choice of an expression system for the production of any recombinant protein is critical, particularly if the protein contains conformational epitopes stabilized by multiple disulfide bonds. Conventionally, Escherichia coli is considered unsuitable for the expression of such structured antigens because of its reducing cytoplasmic environment (18). For that reason, many P. vivax antigens containing complex tertiary-domain structures have been expressed in eukaryotic systems, such as yeast (14,15,16) and baculovirus (9, 22). Efforts have been under way to develop an E. coli strain with an oxidative internal environment. One such modified E. coli strain (Origami) was recently reported to allow disulfide bond formation of recombinant proteins expressed in its cytoplasm (2). Using this strain of E. coli, we report the production of a soluble P. vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42), a malaria vaccine candidate that requires the formation of multiple disulfide bonds for correct folding.MSP-1 is found...

Antigenicity, Immunogenicity, and Protective Efficacy of Plasmodium Vivax Msp1 Pv200l: A Potential Malaria Vaccine Subunit

“…4 Because antibodies specific to this protein, particularly to the C-terminal region (42-kD and 19-kD subfragments), have been shown to block parasite invasion in vitro [5][6][7] and to induce protective immunity in animal models, [8][9][10][11][12][13] this MSP-1 fragment has long been considered a major vaccine candidate. 12,14,15 Limited effort has been invested in the definition and testing of other MSP-1 fragments as potential vaccine candidates.…”

The Plasmodium vivax Merozoite Surface Protein 1 Paralog Is a Novel Erythrocyte-Binding Ligand of P. vivax

“…PvMSP1-19, the C-terminal region of PvMSP1, is a core-binding region for erythrocytes (24). Antibodies against the C terminus of PvMSP1 in P. vivax-immune humans and immunized animals play a major role in the parasite inhibitory response (15,22,25). The paralog of pvmsp1, named pvmsp1p (PVX_099975), is highly conserved among worldwide isolates (26).…”

Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

“…According to Bessette et al (5), a higher yield of oxidized proteins can be obtained in the cytoplasm of the TRXB gor double mutant (gor encodes glutathione reductase). The expression of proteins by the pET-32b expression vector in E. coli Origami (DE3) has been successfully achieved for plant, bacterial, and human proteins (3,19,20,29,37). However, to our knowledge, no peptides containing two disulfide bridges have been expressed in the E. coli Origami/pET 32b system.…”