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

Ranheim, Todd S.; Mathis, Pamela K.; Joelsson, Daniel B.; Smith, Mary Ellen; Campbell, Kendall M.; Lucas, Gwendolyn; Barmat, Scott L.; Melissen, Erin; Benz, R.; Lewis, John A.; Chen, Jie; Schofield, Timothy L.; Sitrin, Robert D.; Hennessey, John P.

doi:10.1016/j.jviromet.2005.08.013

Cited by 49 publications

(35 citation statements)

References 13 publications

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“…A similar study using the SYBR green detection method was effective at estimating viral loads in specimens infected with a diverse range of genotypes, including P[8], G9 and P[4], G2 . Other assays were designed to detect only specific genotypes or were limited in the number of genotypes tested [Schwarz et al, 2002;Pang et al, 2004;Logan et al, 2006;Ranheim et al, 2006;Ray et al, 2006]. Additionally, the region of NSP3 selected for primer and probe design [Pang et al, 2004] is believed to be an optimal target because it may be more highly conserved than regions of other genome segments chosen as targets, including VP6 and VP4, that have been used [Schwarz et al, 2002;Pang et al, 2004;Logan et al, 2006;Min et al, 2006;Ray et al, 2006].…”

Section: Discussionmentioning

confidence: 99%

Enhancement of detection and quantification of rotavirus in stool using a modified real‐time RT‐PCR assay

Freeman

Kerin

Hull

et al. 2008

Journal of Medical Virology

130

104

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A sensitive and specific real-time RT-PCR assay to detect rotavirus in stool samples was optimized and validated using a wide range of rotavirus genotypes. The target of the original TaqMan(R) assay is an 87 bp fragment of the highly conserved non-structural protein 3 (NSP3) gene. Here we modified the original assay by introducing degeneracy into the forward primer to account for sequence variation between rotavirus genotypes, added four nucleotides at the 3' end of the reverse primer to reduce its stability, and modified the probe label. Amplification and detection conditions were optimized using purified dsRNA from two cultivated strains. The limit of detection of the modified assay was calculated to be approximately 44 genome copies per reaction. To validate the reactivity of the assay, 103 archived RNAs that had been extracted from stools and genotyped during routine U.S. surveillance were tested. Samples were selected to represent both rare and common genotypes that have been detected in U.S. children. Nine genotypes known to be circulating in the United States were detected by the real-time assay demonstrating broad reactivity. In addition, other enteric viruses were not detected demonstrating that the assay is specific for rotavirus and does not cross-react with other viruses potentially present in stool samples. This real-time assay is an important addition to the arsenal of molecular tools available to quickly identify rotavirus in stool samples during routine surveillance.

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Section: Discussionmentioning

confidence: 99%

Enhancement of detection and quantification of rotavirus in stool using a modified real‐time RT‐PCR assay

Freeman

Kerin

Hull

et al. 2008

Journal of Medical Virology

130

104

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“…47 Briefly, 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 uses primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard.…”

Section: Methodsmentioning

confidence: 99%

“…The assay was validated for non-interference from all formulation ingredients used in this study. The limit of quantification using in-house standards used in this study was determined as described earlier 47 and was found to be Log 10 5.70 IU/ mL giving quantitation range of 2 log. The lyophilized formulations were tested for potency under different storage conditions as indicated.…”

Section: Methodsmentioning

confidence: 99%

Rational design of heat stable lyophilized rotavirus vaccine formulations

Madhu

Sikriwal

Sharma

et al. 2018

Human Vaccines & Immunotherapeutics

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To develop a safe and efficacious heat-stable rotavirus vaccine, new lyophilized formulations were developed using rotavirus serotypes constituting RotaTeq®. A series of formulation compositions, differing in buffering agents, bulking agents, cryoprotectants, amino acids and divalent cations, were screened for their ability to provide stability to rotavirus serotypes during lyophilization and when stored under elevated temperatures for extended periods. Lead formulations and lyophilization cycles were further optimized. Stability profiles of thus optimized formulations showed their ability to retain the potency of rotavirus for > 36 months at 5°C, 20 months at 37°C, and 7 months at 45°C. The heat-stable lyophilized rotavirus formulations developed met the all critical quality attributes for appearance, heat-stability during storage, moisture content as well as pH, viability and stability after reconstitution and has great potential to be used as vaccine candidates for improving access in low-income countries.

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“…Th erefore, in order to ensure an effi cient immune response, accurate and rapid determination of the rotaviral titer is important in the process of developing a rotavirus vaccine. Various methods have been developed to measure the rotavirus titers, including the plaque assay (Ranheim et al, 2006), the TCID 50 (virus concentration, at which 50% of infected cultures show substantial cytopathology; based on end-point serial dilutions) assay, the reverse transcription-quantitative PCR (RT-qPCR) (Mukhopadhya et al, 2013) and lgCCID 50 -ELISA. The potency of live virus vaccines has traditionally been determined with the plaque and TCID 50 assays, which quantify the infectious virus (Gaush and Smith, 1968;Smith et al, 1979;Sood et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Detecting rotavirus vaccine by these methods will take several days or even one week for the cytopathology to become apparent. Ranheim et al (2006) established a sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq®, Merck KgaA, Darmstadt, Germany) as an alternative to classical potency assays. Potency was determined by assaying cellbased viral replication, followed by RT-qPCR analysis.…”

Section: Introductionmentioning

confidence: 99%

Establishment of indirect immunofluorescence assay for rotavirus

Jiang¹,

Zhang²,

Liu³

et al. 2016

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Summary. -Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a signifi cant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most eff ective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofl uorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. Th e activating concentration of trypsin was 1 μg/ml for healthy monolayers of MA104 cells at 100% confl uence. Aft er incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fl uorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID 50 -ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certifi cation of rotavirus vaccine production.Keywords: rotaviruses; diarrhea and dehydration; titration; indirect immunofl uorescence assay; lgCCID 50 -ELISA * E-mail: 732450624@qq.com; phone: +0086-18743024507. Abbreviations: CPE = cytopathic eff ect; CV = coeffi cient of variation; FCFU = fl uorescent cell forming units; IFA = indirect immunofl uorescence assay; lgCCID 50 -ELISA = log 50% cell culture infectious dose-ELISA; LLR = Lanzhou lamb rotavirus

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

Cited by 49 publications

References 13 publications

Enhancement of detection and quantification of rotavirus in stool using a modified real‐time RT‐PCR assay

Enhancement of detection and quantification of rotavirus in stool using a modified real‐time RT‐PCR assay

Rational design of heat stable lyophilized rotavirus vaccine formulations

Establishment of indirect immunofluorescence assay for rotavirus

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