Molecular Biotechnology

, Volume 40, Issue 1, pp 47–57 | Cite as

A TaqMan® Reverse Transcription Polymerase Chain Reaction (RT-PCR) In Vitro Potency Assay for Plasmid-based Vaccine Products

  • Rohit Mahajan
  • Beth Feher
  • Basil Jones
  • Doug Jones
  • Lana Marjerison
  • Mindy Sam
  • Jukka Hartikka
  • Mary Wloch
  • Peggy Lalor
  • David Kaslow
  • Keith Hall
  • Alain Rolland
Research

Abstract

A TaqMan®-based reverse transcription polymerase chain reaction (RT-PCR) assay has been developed as an in vitro potency assay to measure the most immediate biological activity of plasmid DNA (pDNA)-based products. The assay measures transgene-specific messenger RNA (mRNA) from cultured cells transfected with VCL-CB01, a bivalent pDNA-based human cytomegalovirus (CMV) vaccine. The forward and reverse primers have been designed to make the RT-PCR reaction selective for plasmid-derived mRNA and to allow discrimination of expression levels of individual plasmids in a multivalent pDNA vaccine. The relative potency of a vaccine lot is assessed by transfecting reference and test samples into cultured cells in parallel and analyzing total RNA from the cells by RT-PCR. Statistical analysis of dose response data from reference material supports a parallel-line model for calculating relative potency. Preliminary data demonstrate the ability of this assay to distinguish product potencies at 50, 75, 150, and 200% of the reference material. In addition, forced degradation of pDNA demonstrates that a decrease in relative potency as measured by the RT-PCR assay in vitro correlates well with a decrease in CMV DNA vaccine-mediated humoral immune responses in mice injected with the same material.

Keywords

Plasmid-based vaccines RT-PCR Potency assay pDNA Real time PCR 

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Copyright information

© Humana Press 2008

Authors and Affiliations

  • Rohit Mahajan
    • 1
  • Beth Feher
    • 1
  • Basil Jones
    • 1
  • Doug Jones
    • 1
  • Lana Marjerison
    • 1
  • Mindy Sam
    • 1
  • Jukka Hartikka
    • 1
  • Mary Wloch
    • 1
  • Peggy Lalor
    • 1
  • David Kaslow
    • 1
    • 2
  • Keith Hall
    • 1
  • Alain Rolland
    • 1
  1. 1.Vical Inc.San DiegoUSA
  2. 2.Infectious Diseases & Vaccines FranchiseMerck Research LaboratoriesNorth WalesUSA

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