Transcriptionally active PCR for antigen identification and vaccine development: In vitro genome-wide screening and in vivo immunogenicity

• Published in: Molecular and biochemical parasitology 2008-03, Vol.158 (1), p.32-45
• Main Authors: Regis, David P., Dobaño, Carlota, Quiñones-Olson, Paola, Liang, Xiaowu, Graber, Norma L., Stefaniak, Maureen E., Campo, Joseph J., Carucci, Daniel J., Roth, David A., He, Huaping, Felgner, Philip L., Doolan, Denise L.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Protozoan - blood; Antigen; Antigens, Protozoan - genetics; Antigens, Protozoan - immunology; Cytokines - biosynthesis; Female; Genome; Malaria; Malaria Vaccines - genetics; Malaria Vaccines - immunology; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; PCR; Plasmodium; Plasmodium falciparum; Plasmodium falciparum - genetics; Plasmodium falciparum - immunology; Plasmodium yoelii - genetics; Plasmodium yoelii - immunology; Polymerase Chain Reaction - methods; Screening; Spleen - immunology; T-Lymphocytes - immunology; Vaccines, DNA - genetics; Vaccines, DNA - immunology
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2007.11.009

We have evaluated a technology called transcriptionally active PCR (TAP) for high throughput identification and prioritization of novel target antigens from genomic sequence data using the Plasmodium parasite, the causative agent of malaria, as a model. First, we adapted the TAP technology for the highly AT-rich Plasmodium genome, using well-characterized P. falciparum and P. yoelii antigens and a small panel of uncharacterized open reading frames from the P. falciparum genome sequence database. We demonstrated that TAP fragments encoding six well-characterized P. falciparum antigens and five well-characterized P. yoelii antigens could be amplified in an equivalent manner from both plasmid DNA and genomic DNA templates, and that uncharacterized open reading frames could also be amplified from genomic DNA template. Second, we showed that the in vitro expression of the TAP fragments was equivalent or superior to that of supercoiled plasmid DNA encoding the same antigen. Third, we evaluated the in vivo immunogenicity of TAP fragments encoding a subset of the model P. falciparum and P. yoelii antigens. We found that antigen-specific antibody and cellular immune responses induced by the TAP fragments in mice were equivalent or superior to those induced by the corresponding plasmid DNA vaccines. Finally, we developed and demonstrated proof-of-principle for an in vitro humoral immunoscreening assay for down-selection of novel target antigens. These data support the potential of a TAP approach for rapid high throughput functional screening and identification of potential candidate vaccine antigens from genomic sequence data.