Ensuring the safety of vaccine cell substrates by massively parallel sequencing of the transcriptome
Introduction
An essential component of vaccine safety is ensuring that the cell substrates and virus seeds are free of adventitious viruses and other extraneous agents. Until recently the main method of analysis has involved in vitro and in vivo infectivity assays and specific PCR analyses. The recent use of massively parallel sequencing, coupled to an interpretive algorithm (MP-Seq), to screen vaccine cell substrates [1] has highlighted the value of this new technology as, an unbiased and comprehensive method to identify viruses and other adventitious agents, without prior knowledge of the nature of those agents. The discovery of contaminating porcine circovirus in vaccines by massively parallel sequencing has reinforced the value of this approach [2].
We have developed massively parallel sequencing methods to detect and characterise viruses in the supernatants from cell substrates, and virus seed stocks, and demonstrated their utility in identifying a new virus in raw materials [1]. Cells used to produce vaccines may harbour novel viruses whose sequences are unknown or, latent viruses that are only expressed following stress to the cells. In these cases, deep sequencing of the cellular transcriptome will record latency associated transcripts or, viral transforming gene sequences, as well as mRNAs associated with replicating viruses. The challenge of this technology is to identify viral transcripts amid a vast background of cellular sequences. The analysis must exclude “false hits” that result from cellular sequences that have been captured by viruses during evolution or following de novo recombination. The key to achieving this is the development of a robust algorithmic method that excludes false hits and enables the identification of viruses, in the absence of cellular genomic information, and without prior knowledge of the specific virus sequence.
Here we describe the screening of two key cell lines, Vero (ATCC CCL-81) and Trichoplusi ni (High Five™, Invitrogen), for adventitious viral agents by MP-Seq. Through these studies, we have excluded the presence of porcine circovirus and other adventitious agents in this Vero cell bank but have demonstrated the expression of a full length betaretrovirus in these cells. This retrovirus may be of importance in the production of certain vaccines, particularly those able to pseudotype retroviruses.
The combination of baculovirus vectors and insect cell substrates is proving to be a valuable system for the generation of viral like particle and sub-unit vaccines. However, one of the unusual features of insect cells is the wide range of viruses that may be latent in these cells, with contaminating viruses being induced into replication after superinfection with a baculovirus vector. In this study, we demonstrate the detection of a contaminating, latent nodavirus and identify an expressed errantivirus genome in the T. ni cell line.
As a further test of the ability of the MP-Seq method to detect novel viruses, we show that even after removal of the specific nodavirus sequence from our curated viral database, we maintain the ability to detect the virus through its close relationship to other viruses. Collectively these results demonstrate the power of MP-Seq and its application to adventitious agent screening.
Section snippets
High Five cells
A T. ni cell line (BTI-TN-5B1-4 “High Five™”; Invitrogen), known to contain a sub-clonal infection with an alphanodavirus [3], was cultured in Grace's medium containing 10% Foetal bovine serum (FBS; Gibco; Life Technologies) at 27 °C. After splitting the cells, one half of the cells were seeded into fresh medium at 27 °C while the remaining cells were heat shocked at 37 °C for 24 h then cultured for a further 48 h at 27 °C. The heat shocked cells and control cells were washed 3 times to remove serum
Development of PCV-specific assays
A step-wise approach to investigate the presence of PCV in Vero cells was developed, aimed at excluding false positives resulting from residual PCV sequences present in porcine trypsin. In the first step, PCV-1 and PCV-2 specific PCR assays were developed to detect fragments of PCV. In the event of detecting positives by PCR, rolling circle amplification, was employed to demonstrate whether the signals were due to residual genomic fragments or represented potentially infectious virions (i.e.
Discussion
The introduction of new cell substrates into vaccine manufacture, like potentially tumourogenic MDCK cells, has prompted the need for comprehensive analysis to exclude the presence of oncogenic viruses [4], [9]. At the same time a renaissance in virus discovery has begun with the advent of massively parallel sequencing and this method revealed that some vaccines contained contaminants that had previously gone undetected [2]. Since massively parallel sequencing methods do not depend on
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