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.

Clinical trial in healthy malaria-naïve adults to evaluate the safety, tolerability, immunogenicity and efficacy of MuStDO5, a five-gene, sporozoite/hepatic stagePlasmodium falciparumDNA vaccine combined with escalating dose human GM-CSF DNA

“…The rationale for multiple antigens was based on the genetic restriction of adaptive cell-mediated immunity: since each malaria antigen provides only limited numbers of strongly-binding class I epitopes for the diverse MHC antigens of the human population, the combination of multiple protective malaria proteins is likely required to achieve broad population coverage 48 . In prior non-clinical studies, we have shown that multivalent DNA vaccines comprising plasmid mixtures can be immunogenic in mice and NHP without significant interference, 16 , 45 , 49 , 73 although plasmids encoding certain malaria proteins could theoretically suppress the immunogenicity of other plasmids if included in the mixture, 50 as shown in vitro 51 …”

Protection of Rhesus Monkeys by a DNA Prime/Poxvirus Boost Malaria Vaccine Depends on Optimal DNA Priming and Inclusion of Blood Stage Antigens

“…We do not pretend to understand these phenomena. Previously, inhibition between DNA vaccines has been described when the vaccines were given together in the same syringe, and was thought to be due to competition between plasmids within mammalian cells [61]–[63]. To avoid such competition, we administered each of our DNA vaccine antigens into muscles on different limbs.…”

Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α)

“…Vaccines that used plasmid DNA (pDNA) as a physical vector encoding an antigen of interest provided a promising method for preventing diseases such as HIV-AIDS, Ebola, malaria, and cancer (Bacon and Sedegah, 2007;Boyer et al, 2000;Martin et al, 2006) introduced in 1990 when purified DNA was injected intramuscular which yielded both gene expression and biological activity (Jiao et al, 1992;Wolff et al, 1990). Compared with protein vaccines where microgram quantities of the antigen are required, milligram amounts of pure, super coiled pDNA are needed in order to initiate cellular and humoral immune responses (Tavel et al, 2007).…”