Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq).
A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines.
Ranheim
Todd
T
Bioprocess & Bioanalytical Research, Merck Research Laboratories, Merck & Co., Inc., WP75-300, West Point, PA 19486, USA. todd_ranheim@merck.com
Mathis
Pamela K
PK
Joelsson
Daniel B
DB
Smith
Mary Ellen
ME
Campbell
Kathryn M
KM
Lucas
Gwendolyn
G
Barmat
Scott
S
Melissen
Erin
E
Benz
Robert
R
Lewis
John A
JA
Chen
Jie
J
Schofield
Timothy
T
Sitrin
Robert D
RD
Hennessey
John P
JP
D000818:Animals; D002522:Chlorocebus aethiops; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000502:physiology; D022243:Rotavirus Vaccines / Q000235:genetics; D014613:Vaccines, Attenuated / Q000235:genetics; D014709:Vero Cells; D010948:Viral Plaque Assay; D014779:Virus Replication
D012367:RNA, Viral; C492535:RotaTeq; D022243:Rotavirus Vaccines; D014613:Vaccines, Attenuated