title: Reduced production of RNA transcripts from individual DNA plasmids given in a multivalent DNA vaccine formula.
abstract: We conducted transient transfection studies using two DNA vaccines constructs encoding two Plasmodium falciparum surface proteins, PfCSP and PfSSP2, and UM449 melanoma cells to determine transcription and translation efficiencies. Plasmids were transfected individually or in combination with an empty control plasmid with and without a functional CMV IE promoter. Western blot analysis using NSF1, a monoclonal antibody specific for PfCSP, and UM449 cell lysate revealed an abrogation in expression of PfCSP when a plasmid carrying the Pfcsp gene was cotransfected with an empty control plasmid with a functional CMV IE promoter. When a control plasmid without a functional CMV IE promoter was substituted in the expression study, normal levels of PfCSP were detected by Western blot. Total RNA was isolated following transfection and reverse transcriptase quantitative (RTQ)-PCR was performed. Levels of Pfcsp and Pfssp2 transcripts decreased significantly when cotransfected with a control plasmid containing a functional CMV IE promoter while transcript levels of Pfcsp and Pfssp2 were significantly higher in cells cotransfected with a control plasmid without a functional CMV IE promoter. The presence of multiple copies of a functional CMV IE promoter leads to a decrease in expression of malaria antigens present in a multivalent vaccine mixture when transfected in vitro.
authors.affiliation: Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, Maryland, USA. bacon@nmrcd.med.navy.mil
authors.forename: David J
authors.initials: DJ
authors.lastname: Bacon
authors.forename: Martha
authors.initials: M
authors.lastname: Sedegah
mesh_terms: D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017780:Malaria Vaccines / Q000235:genetics* / Q000276:immunology; D016778:Malaria, Falciparum / Q000276:immunology* / Q000517:prevention & control; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology*; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology
chemical_list: D017780:Malaria Vaccines; C487838:PfCSP DNA vaccine; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D019444:Vaccines, DNA