Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq^®)

Abstract

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 24 h. 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.

Introduction

Worldwide, rotavirus is the primary viral etiologic agent of serious diarrheal illness in infants and young children <2 years old (Kapikian et al., 2001) and is associated with an estimated 111 million episodes of gastroenteritis and 600,000 deaths per year (Parashar et al., 2003). In the United States it is estimated that rotavirus infections are responsible for 50,000 hospitalizations and $1 billion in costs to society annually (Tucker et al., 1998). The immunogenicity, safety and efficacy of a live human-bovine (WC3) reassortant rotavirus vaccine has been demonstrated in previous clinical trials (Clark et al., 1996a, Clark et al., 1996b, Clark et al., 2003, Clark et al., 2004) and is being evaluated in a large (>70,000 subject), placebo-controlled clinical trial.

Rotaviruses belong to the Reovirus genus in the Reoviridae family of viruses. Rotaviruses are large, nonenveloped icosahedral particles composed of three concentric protein layers surrounding 11 genome segments of double-stranded RNA (Estes, 2001). The outer layer is composed of the VP7 and VP4 proteins which are the main antigenic determinants of the virus (Estes, 2001). The VP4 protein is the viral attachment protein and rotavirus infectivity is enhanced by proteolytic cleavage of VP4 into two cleavage products that remain associated with the particle (Ciarlet and Estes, 2002).

The segmented nature of the rotavirus genome allows for genetic reassortment to occur during infections of humans, animals, or cultured cells with multiple rotavirus strains. Genetic reassortment was used to create the five human-bovine reassortant strains in the pentavalent vaccine (RotaTeq^®) currently under development by Merck & Co., Inc. The four human rotavirus G serotypes contained in the vaccine, WI79-9, SC2-9, WI78-8, and BrB-9 (designated G1–G4 for simplicity), are known to cause >80% of all rotavirus disease in the United States, and >85% of rotavirus disease worldwide and the human P serotype in the vaccine, WI79-4 (designated P1), is the most common human serotype associated with rotavirus disease (Clark et al., 1996a, Clark et al., 1996b, Kapikian et al., 2001, Ciarlet and Estes, 2002).

The determination of potency of live virus vaccines has traditionally relied on plaque or TCID₅₀ (virus concentration at which 50% of infected cultures show substantial cytopathology; based on end-point serial dilutions) methods for the quantitation of infectious virus (Gaush and Smith, 1968, Smith et al., 1979, Sood et al., 1995). These assays are typically highly variable, labor intensive, and time consuming, requiring multiple days or even weeks for the appearance of cytopathology and the detection of viral infection. In addition, application of these methods to multivalent vaccines is problematic, requiring the availability of highly specific neutralizing antibodies for each of the components in order to allow specific detection of the infectivity of the individual components. Recently, alternative detection and quantitation techniques have been developed. A combination of reverse transcription (RT) and the polymerase chain reaction (PCR) technique (Heid et al., 1996) have been used in the detection and quantitation of viruses including rotaviruses (Schwarz et al., 2002, Kang et al., 2004), and a recent report has described the application of these techniques in the estimation of infectious measles viruses (Schalk et al., 2004).

In this study, the development and application of a quantitative RT-PCR method for determining the infectivity (i.e. potency) of each of the viral components of the pentavalent rotavirus vaccine, RotaTeq^® is described. The method utilizes primers and probes specific to the VP7 gene (genome segment 9) of each of the rotavirus reassortant strains resulting in highly specific detection of each virus in the presence of the other rotaviruses in the vaccine. The method has proven to be accurate, precise, robust, and amenable to high throughput testing in a quality control laboratory. Important aspects of the development of the assay are presented along with evidence demonstrating correlation with the plaque assay and results from the application of the assay in the manufacture and release of vaccine material.