title: Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.
abstract: The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.
authors.affiliation: Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue, Room 3A4O, Silver Spring, MD 20910-7500, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Witney
authors.forename: D L
authors.initials: DL
authors.lastname: Doolan
authors.forename: R M
authors.initials: RM
authors.lastname: Anthony
authors.forename: W R
authors.initials: WR
authors.lastname: Weiss
authors.forename: S L
authors.initials: SL
authors.lastname: Hoffman
authors.forename: D J
authors.initials: DJ
authors.lastname: Carucci
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004275:DNA, Ribosomal / Q000032:analysis; D004912:Erythrocytes / Q000469:parasitology; D005260:Female; D020459:Genes, rRNA; D007116:Immunization, Passive; D008099:Liver / Q000469:parasitology*; D008288:Malaria / Q000469:parasitology / Q000517:prevention & control*; D017780:Malaria Vaccines / Q000008:administration & dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010967:Plasmodium yoelii / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism; D014611:Vaccination; D019444:Vaccines, DNA / Q000008:administration & dosage
chemical_list: D000911:Antibodies, Monoclonal; D004275:DNA, Ribosomal; D017780:Malaria Vaccines; D012337:RNA, Ribosomal, 18S; D019444:Vaccines, DNA; D019914:Taq Polymerase