Purification, characterization, and immunogenicity of a disulfide cross-linked Plasmodium vivax vaccine candidate antigen, merozoite surface protein 1, expressed in Escherichia coli.
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 > or = 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.
Dutta
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Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500, USA.
Ware
L A
LA
Barbosa
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A
Ockenhouse
C F
CF
Lanar
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DE
D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D017780:Malaria Vaccines / Q000276:immunology*; D016780:Malaria, Vivax / Q000517:prevention & control*; D008353:Mannitol / Q000031:analogs & derivatives; D020066:Merozoite Surface Protein 1 / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009829:Oleic Acids; D010966:Plasmodium vivax / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014611:Vaccination; D014614:Vaccines, Synthetic / Q000276:immunology
D000276:Adjuvants, Immunologic; D000913:Antibodies, Protozoan; D003432:Cross-Linking Reagents; D004220:Disulfides; D017780:Malaria Vaccines; D020066:Merozoite Surface Protein 1; D009829:Oleic Acids; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C001681:mannide monooleate; D008353:Mannitol