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Infection and Immunity, September 2001, p. 5464-5470, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5464-5470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Sheetij Dutta, Lisa A. Ware, Arnoldo Barbosa, Christian F. Ockenhouse, and David E. Lanar*

Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500

Received 26 March 2001/Returned for modification 24 May 2001/Accepted 14 June 2001

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.

* Corresponding author. Mailing address: Department of Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910-7500. Phone: (301) 319-9003. Fax: (301) 319-7358. E-mail: david.lanar{at}na.amedd.army.mil.

Infection and Immunity, September 2001, p. 5464-5470, Vol. 69, No. 9
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.9.5464-5470.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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