Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line

Azizi, Ali; Aidoo, Francisca; Gisonni-Lex, Lucy; McNeil, Bryan

doi:10.1016/j.jbiotec.2013.10.002

Cited by 4 publications

(6 citation statements)

References 29 publications

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“…AV529-19 is a Vero cell line specifically engineered to express the coding regions of transgenes HSV-1 UL5 and UL29; it is used for the propagation of a candidate herpesvirus type II vaccine with defective UL5 and UL29 coding regions. 13 The first approach was to develop a qPCR-based method to assess UL5 and UL29 gene copy number. This assay is, based on a relative quantitation that involves an in-house reference standard from which reportable values for UL5 and UL29 gene copy numbers are extrapolated.…”

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

“…However, as the Vero cell line was not fully sequenced at the time, construction of a test reference standard without significant optimization was difficult. 13 Therefore, a second molecular method was developed, using digital PCR (dPCR) technology to demonstrate the stability of the transgene copy number in the AV529-19 cell line. 13 This high throughput dPCR-based approach did not require any test reference standard, and demonstrated high precision and accuracy.…”

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

“…13 Therefore, a second molecular method was developed, using digital PCR (dPCR) technology to demonstrate the stability of the transgene copy number in the AV529-19 cell line. 13 This high throughput dPCR-based approach did not require any test reference standard, and demonstrated high precision and accuracy. dPCR involves partitioning each test sample into a large number of individual reactions, with reaction partitions that received a target sequence counted as positive and those reaction partitions that did not receive a target molecule counted as negative.…”

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

“…The developed method was able to resolve the issues associated with the traditional qPCR-based approach, and confirmed the stability of UL5 and UL29 transgenes in the Vero MCB and ECB. 13 High throughput sequencing approach for evaluation of genetic stability in vaccine cell lines…”

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

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New approaches for characterization of the genetic stability of vaccine cell lines

Gisonni-Lex

Azizi

2017

Human Vaccines & Immunotherapeutics

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The genetic stability of cell lines is a critical analytical attribute required to demonstrate the quality of cells over time. During cell passage, mutations can arise in the genomic DNA, potentially leading to changes in the final vaccine product. The identity and integrity of master cell banks, extended cell banks, complementing cell lines or recombinant cell lines expressing transgenes has to be tested throughout the production process by the vaccine manufacturer. Over the past few years, the traditional methods for evaluation of genetic stability have been replaced with molecular approaches including quantitative PCR, digital PCR and high throughput sequencing. However, these molecular-based approaches are used in research laboratories and not within a GMP-compliant environment. In this article, we briefly discuss some opportunities and challenges in characterization of the genetic stability of vaccine cell lines with these molecular-based approaches.

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Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

Section: Stability Of Transgenes In Complementing Cell Linesmentioning

confidence: 99%

See 2 more Smart Citations

New approaches for characterization of the genetic stability of vaccine cell lines

Gisonni-Lex

Azizi

2017

Human Vaccines & Immunotherapeutics

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“…There is considerable variety of cell lines with these features; however, in this review, in most of the studies cited (Table 1), Vero cells were used. These cells are widely used in antiviral assays because the lytic cycle of several viruses can be reproduced [79,80], among these HSV [81].…”

Section: Medicinal Plant Extracts With Anti Herpetic Activitymentioning

confidence: 99%

Antiherpetic Plants: A Review of Active Extracts, Isolated Compounds, and Bioassays

Silva-Mares

Torres‐López

Rivas-Galindo

2016

Natural Product Communications

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Herpes simplex is a disease that is widely distributed throughout the world. It is caused by herpes simplex virus type 1 (HSV-1) and simplex virus type 2 (HSV-2). The drugs of choice for treatment are acyclovir (ACV), Penciclovir (PCV) and other guanine analogues, which have the same mechanism of action. However, due to the constant increase of ACV-resistant strains in immunocompromised patients, it is necessary to find new treatment alternatives. It has been shown that natural products are a good alternative for the treatment of these diseases as well as being an excellent source of compounds with anti-herpetic activity, which may be useful for the development of new drugs and act through a mechanism of action different from ACV and PCV. This paper compiles reports on extracts and compounds isolated from plants that have anti-herpetic activity. We present an analysis of the solvents most widely used for extraction from plants as well as cells and commonly used methods for evaluating cytotoxic and anti-herpetic activity. Families that have a higher number of plants with anti-herpetic activity are evaluated, and we also highlight the importance of studies of mechanisms of action of extracts and compounds with anti-herpetic activity.

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Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate

Fradin

Pędyczak

Carpick

2018

Journal of Pharmaceutical Sciences

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Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line

Cited by 4 publications

References 29 publications

New approaches for characterization of the genetic stability of vaccine cell lines

New approaches for characterization of the genetic stability of vaccine cell lines

Antiherpetic Plants: A Review of Active Extracts, Isolated Compounds, and Bioassays

Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate

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