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Infection and Immunity, March 2004, p. 1594-1602, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1594-1602.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

Diana Haddad,1,{dagger} Erika Bilcikova,1 Adam A. Witney,2 Jane M. Carlton,3 Charles E. White,4 Peter L. Blair,1 Rana Chattopadhyay,1 Joshua Russell,1 Esteban Abot,1 Yupin Charoenvit,1 Joao C. Aguiar,1 Daniel J. Carucci,1 and Walter R. Weiss1*

Naval Medical Research Center and,1 Walter Reed Army Institute of Research, Silver Spring, and,4 Institute for Genomic Research, Rockville, Maryland, and,3 St. George's Hospital Medical School, London, United Kingdom2

Received 31 July 2003/ Returned for modification 29 September 2003/ Accepted 18 November 2003

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

* Corresponding author. Mailing address: Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910. Phone: (310) 319- 7563. Fax: (301) 319-7545. E-mail: weissw{at}nmrc.navy.mil.

Editor: W. A. Petri, Jr.

{dagger} Present address: Department of Molecular Microbiology and Immunology, The Johns Hopkins School of Public Health, Baltimore, MD 21205.

Infection and Immunity, March 2004, p. 1594-1602, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1594-1602.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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