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 encodingtwo Plasmodium falciparum surface proteins, PfCSP and PfSSP2, and UM449 melanoma cells todetermine transcription and translation efficiencies. Plasmids were transfected individually or incombination 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 celllysate revealed an abrogation in expression of PfCSP when a plasmid carrying the Pfcsp genewas co-transfected with an empty control plasmid with a functional CMV IE promoter. When acontrol 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 followingtransfection and reverse transcriptase quantitative (RTQ)-PCR was performed. Levels of Pfcspand Pfssp2 transcripts decreased significantly when co-transfected with a control plasmidcontaining a functional CMV IE promoter while transcript levels of Pfcsp and Pfssp2 weresignificantly higher in cells co-transfected with a control plasmid without a functional CMV IEpromoter. The presence of multiple copies of a functional CMV IE promoter leads to a decreasein expression of malaria antigens present in a multivalent vaccine mixture when transfected invitro.