Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay

Schalk, J.A.C.; Vries, Claudette G. J. C. A. de; Jongen, Peter M.J.M.

doi:10.1016/j.biologicals.2005.01.001

Cited by 23 publications

(15 citation statements)

References 13 publications

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“…There are several challenges with conventional in-vitro assays (plaque or CPE) to measure the titer of live attenuated or defective viral-based vaccines [4,6]. The traditional assays are usually laborious and sometimes hard to interpret.…”

Section: Discussionmentioning

confidence: 99%

“…After 24 hours, Vero cells were lysed and the lysates were measured by RT-qPCR to quantify viral replication. In another study, Schalk et al, [4] developed a rapid assay for the measurement of infectivity-potency in MMR trivalent vaccines based on a qPCR infectivity assay. The assay was able to demonstrate the potency of mumps and measles viruses within a period of 2 days.…”

Section: Introductionmentioning

confidence: 99%

“…The assay was able to demonstrate the potency of mumps and measles viruses within a period of 2 days. Since rubella virus replicates slower than measles and mumps, the potency estimation for rubella virus was PCR-based assays as end-points since a plaque assay for measles and rubella virus usually takes 9 days [4]. This period of time for detection of mumps virus in cell line is 6 days.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach

et al. 2013

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BackgroundOne of the critical tasks in analytical testing is to monitor and assign the infectivity or potency of viral based vaccines from process development to production of final clinical lots. In this study, a high throughput RT-qPCR based approach was developed to evaluate the infectious titre in a replication-defective HSV-2 candidate vaccine, called HSV529. This assay is a combination of viral propagation and quantitative RT-PCR which measures the amount of RNA in infected cells after incubation with test samples.ResultsThe relative infectious titre of HSV529 candidate vaccine was determined by a RT-qPCR method targeting HSV-2 gD2 gene. The data were analyzed using the parallel-line analysis as described in the European Pharmacopoeia 8th edition. The stability of HSV529 test samples were also investigated in a concordance study between RT-qPCR infectivity assay and a classical plaque assays. A suitable correlation was determined between both assays using an identical sample set in both assays. The RT-qPCR infectivity assay was further characterized by evaluating the intermediate precision and accuracy. The coefficient of variation from the six independent assays was less than 10%. The accuracy of each of the assay was also evaluated in the range of 92.91% to 120.57%.ConclusionsOur data demonstrate that the developed RT-qPCR infectivity assay is a rapid high throughput approach to quantify the infectious titer or potency of live attenuated or defective viral-based vaccines, an attribute which is associated with product quality.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach

et al. 2013

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“…The reaction mixture (20 μl) consisted of medium Master HybProbe, 50 ng of total RNA, 3.25 mM Mn[OAc] 2 , 0.5 μM of each primer and 0.2 μM of each probe. The primers and probes used were as described previously [30] and are listed in Table 1. As a negative controls water was run with every PCR.…”

Section: Methodsmentioning

confidence: 99%

Expression of TNF-α, OPG, IL-1β and the presence of the measles virus RNA in the stapes of the patients with otosclerosis

Potocka-Bakłażec

Sakowicz-Burkiewicz

Kuczkowski

et al. 2014

Eur Arch Otorhinolaryngol

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Persistent measles virus infections play a crucial role in the pathomechanism of otosclerosis. The study was undertaken to investigate the role of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and osteoprotegerin (OPG) in otosclerotic bone remodeling and to assess the relation of TNF-α, OPG and IL-1β expression levels in otosclerotic stape footplates to the occurrence of measles virus infection. 61 patients with otosclerosis were treated surgically. Thirty-one stapes obtained from cadavers of people, who had died from a sudden cause were used as a control group. The presence of measles virus RNA and the expression levels of TNF-α, IL-1β and OPG in otosclerotic foci were assessed using one-step RT-PCR. The presence of measles virus RNA was noted in 80.3 % of otosclerotic stapes (49 out of 61) and 9.7 % of normal tissues (3 out of 31). Transcript of TNF-α, IL-1β and OPG was detected in 40, 46 and 18 virus-positive stapes, respectively. The transcript level of TNF-α and IL-1β was significantly higher in otosclerotic tissues comparing to normal tissue. The OPG expression level was significantly lower in otosclerotic tissues comparing to controls. The presence of measles virus RNA in the stapes may indicate its role in the pathogenesis of otosclerosis. The presence of TNF-α and IL-1β mRNA in the virus-positive stapes could be the result of viral antigen stimulation and may be a marker of inflammation the otosclerotic focus. The lack of OPG mRNA and the presence of TNF-α and IL-1β mRNA in the majority of otosclerotic tissues reflect the bone remodeling process occurring in the stapes.

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“…While the increased sensitivity of PCR-based assays allows for a more rapid determination of viral efficacy, the specificity of the PCR reagents allows for the determination of infectivity of individual components of a multivalent viral vaccine. Viral titers of new lots of product have been determined either by PCR measurement of total viral DNA in infected tissue culture cells [10,11] or by quantitation of virusspecific RNA produced upon infection [12][13][14][15][16]. The development of these new methods of determining vaccine potency is supported by the correlation of results with those obtained using traditional methods.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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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.

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Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay

Cited by 23 publications

References 13 publications

Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach

Evaluation of infectious titer in a candidate HSV type 2 vaccine by a quantitative molecular approach

Expression of TNF-α, OPG, IL-1β and the presence of the measles virus RNA in the stapes of the patients with otosclerosis

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

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