A TaqMan reverse transcription polymerase chain reaction (RT-PCR) in vitro potency assay for plasmid-based vaccine products.
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.
Mahajan
Rohit
R
Vical Inc., San Diego, CA 92121, USA. rmahajan@vical.com
Feher
Beth
B
Jones
Basil
B
Jones
Doug
D
Marjerison
Lana
L
Sam
Mindy
M
Hartikka
Jukka
J
Wloch
Mary
M
Lalor
Peggy
P
Kaslow
David
D
Hall
Keith
K
Rolland
Alain
A
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014612:Vaccines / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D014612:Vaccines