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

Abstract

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.The year 2002 saw the publication of the genomic sequences of the human malaria parasite Plasmodium falciparum (12) and the rodent parasite Plasmodium yoelii yoelii (5). The hope is that this information will bring insights into parasite biology and lead to the development of new vaccines and drugs. However, novel research approaches are required to efficiently study the thousands of genes. This paper describes the development of a high-throughput technique for the identification of vaccine target antigens among newly annotated malaria genes. Our method rapidly produces large numbers of DNA vaccines carrying P. y. yoelii exons and measures their ability to reduce parasite load in mice. We call this screening technique the antigen identification method.The novelty and efficiency of the antigen identification method come from a combination of rapid production of DNA vaccines and sensitive measurement of parasite killing. With the annotated P. y. yoelii genomic sequence, we identify P. y. yoelii genes expressed during the sporozoite stage by comparison with expressed sequence tags (ESTs) generated from a cDNA library of P. yoelii sporozoites (20). PCR primers for these sporozoite P. y. yoelii genes are synthesized to be compatible with the Gateway cloning system, which allows rapid production of DNA vaccine plasmids. Mice are immunized with the DNA...

Induction of Strain-Transcending Immunity againstPlasmodium chabaudi adamiMalaria with a Multiepitope DNA Vaccine

“…Twenty of the protective sequences Ͼ50 aa in length encode previously untested hypothetical proteins, and 14 of these proteins showed conservation across several malaria species, suggesting that conserved hypothetical proteins may be contributing to the observed crossprotection. This is analogous to results of Melby et al (39), who identified pools of protective hypothetical proteins within a Leishmania ELI vaccine, and Haddad et al (24), who identified hypothetical proteins in a P. yoelii liver-stage exon DNA vaccine pool. Our next objective is to further segregate the sequences to identify the minimal number of plasmids capable of inducing cross-protection.…”

A genetic approach to thede novoidentification of targets of strain-specific immunity in malaria parasites

“…With the completion of the Plasmodium falciparum Genome Project, bioinformatic approaches are now being used (17). Large numbers of sequences predicted as exons from Plasmodium genomic databases have been screened as DNA vaccine constructs (18).…”

Infection withPlasmodium bergheiBoosts Antibody Responses Primed by a DNA Vaccine Encoding Gametocyte Antigen Pbs48/45

“…Thus, the entire forward and reverse primer sequences used here in the amplification of the Pbs48/45 gene were 5Ј-GCAGG CTCCACCATGAATGAGTATGTTTCTCCAGATGAA-3Ј and 5Ј-AGAAAG CTGGGTTTACATAAAACCAGTTATTTTATCCAT-3Ј, respectively. PCR amplification and Gateway cloning was carried out using conditions described previously (14). A fragment of approximately 1.2 kb was obtained by PCR amplification for 35 cycles consisting of 94°C for 30 s, 52°C for 60 s, and 68°C for 2 min.…”

“…Entry and DV1020 clones were analyzed by PCR and sequencing using vector-specific primers (14). Plasmid DNA from a confirmed DV1020-Pbs48/45 clone was prepared and purified using an endotoxin-free plasmid purification Giga kit (QIAGEN Inc., Valencia, CA).…”

“…Cloning of the Pbs48/45 fragment into the DNA vaccine vector DV1020 (1) was carried out by means of an intermediary Gateway entry vector (pDONR207) as described before (14). Entry and DV1020 clones were analyzed by PCR and sequencing using vector-specific primers (14).…”