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Infection and Immunity, October 2005, p. 6530-6536, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6530-6536.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of Three Pichia pastoris-Expressed Plasmodium falciparum Merozoite Proteins as a Combination Vaccine against Infection with Blood-Stage Parasites

Dongmei Zhang and Weiqing Pan^*

Department of Etiologic Biology, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, China

Received 13 April 2005/ Returned for modification 19 April 2005/ Accepted 15 June 2005

Because invasion of erythrocytes by Plasmodium falciparum merozoites involves multiple receptor-ligand interactions, it may be necessary to develop a multivalent malaria vaccine that is comprised of distinct parasite ligands. PfAMA-1, PfMSP1, and PfEBA-175 are merozoite proteins that play important roles in invasion. We have constructed a PfCP-2.9 chimeric protein consisting of PfAMA-1 and PfMSP1 and tested it for immunogenicity in animal models and humans. The F2 subdomain of PfEBA-175 (PfEBA-175II F2) was identified as the binding domain for glycophorin A on erythrocytes. In this study, we used the codon frequencies of the yeast Pichia pastoris to redesign and synthesize a gene encoding the F2 domain. We found that the codon-optimized gene was expressed at a high level in P. pastoris as a soluble protein with a yield of about 300 mg/liter. The expressed protein was able to bind normal erythrocytes but not those treated with neuraminidase or trypsin. Moreover, the protein was recognized by the sera of malaria patients and was highly immunogenic in mice, rabbits, and rhesus monkeys. Immunoglobulin G isolated from both immunized rabbits and monkeys inhibited in vitro parasite growth. Immunization of animals with a combination of PfEBA-175II F2 and PfCP-2.9 did not result in antigen (Ag) competition in animals. Moreover, antibodies to both PfEBA-175II F2 and PfCP-2.9, isolated from rabbits immunized with both constructs, inhibited parasite growth in vitro. The combination of high yield, functional folding, antibody inhibition, and lack of Ag competition provides support for inclusion of these merozoite proteins in a combination vaccine against infection with blood-stage parasites.

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* Corresponding author. Mailing address: Department of Etiologic Biology, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, China. Phone: 86-21-25070319. Fax: 86-21-65331272. E-mail: wqpan0912{at}yahoo.com.cn.

Editor: J. F. Urban, Jr.

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Infection and Immunity, October 2005, p. 6530-6536, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6530-6536.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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