Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use.

Janeth Del Carmen Arias Palacios,José Salvador Montaña Lara,Ángela María Londoño Navas,Carlos Alberto Barrero Barreto

doi:10.2174/1874104501711010066

Janeth Del Carmen Arias Palacios, José Salvador Montaña Lara + Show 2 more

Open Access

https://doi.org/10.2174/1874104501711010066

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Abstract

Objectives:Normalize the quantification of residual DNA from Vero cells in the rabies vaccine for use in human VAHV I, by quantitative PCR in real time and the design of primers that amplified, highly repetitive sequences of Cercopithecus aethiops and a constitutive gene according to sequences reported in the GenBank and quantifying the residual DNA in the vaccine VAHV I in three consecutive batches according to the standard set by the World Health Organization.Methods:A real time quantitative method based on SYBR Green chemistry has been applied for the quantification of residual DNA (resDNA) using highly repetitive DNA (Alu) and a housekeeping gene (B-actin) as target sequences.Results:The sensitivity achieved with this white sequence is within the reported limits and who are between 5 and 50 pg. For real time PCR optimization with Alu-p53, different concentrations of MgCl2 (0.5, 0.75, 1.0, 1.25 and 1.5 mm) in combination with three different concentrations of primers (75, 100 and 150nM) were used. pDNA in concentration of 1x107 copies / ul was used as template. Optimal concentrations were 1.25 mM MgCl2 and 100nM primers. To level of detection of 1.53 ng/ul was found for p53-Alu and Alu-Glob and 0.39 ng/ul for B-actin with gDNA curves.Conclusion:Quantification of resDNA of vaccine VAHV I with close-ups of B-actin was normalized. Reached a sensitivity of 30 pg of resDNA/dose VAHV I, with close-ups of B-actin. Found, in three consecutive batches, an amount less than 10 ng/dose, these results suggest that the production process ensures vaccine resDNA removal, meeting international requirements for biological products for use in humans that use continuous cell lines for production.

Highlights

Rabies is widely spread across the world being responsible for around 50.000 human deaths per year
Normalize the quantification of residual DNA from Vero cells in the rabies vaccine for use in human VAHV I, by quantitative PCR in real time and the design of primers that amplified, highly repetitive sequences of Cercopithecus aethiops and a constitutive gene according to sequences reported in the GenBank and quantifying the residual DNA in the vaccine VAHV I in three consecutive batches according to the standard set by the World Health Organization
In three consecutive batches, an amount less than 10 ng/dose, these results suggest that the production process ensures vaccine residual DNA (resDNA) removal, meeting international requirements for biological products for use in humans that use continuous cell lines for production

Summary

Introduction

Rabies is widely spread across the world being responsible for around 50.000 human deaths per year. Kidney epithelial cells isolated from African green monkey (Vero cells) are the cell line of choice for rabies vaccine production. Vero cell line has been used for the production of polio, measles and yellow fever vaccines. During the production of rabies vaccine, vero cells are inoculated with rabies virus, followed by a purificaction step that eliminates cell debris, resulting in virus concentration and inactivation with ®-propiolactone (bpl) [1, 2]. Some researchers consider the presence as innocuous but for some others, the presence of resDNA might pose the risk of: malignant transformations due to the activation of cellular or viral oncogenes, aberrant gene expression resulting from sequence insertions in genomic control regions and production of infectious viral particles through the insertion of viral DNA [3]

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