By Monica Heger
Researchers expecting to demonstrate with next-generation sequencing that live attenuated viral vaccines contain only known viral genomes were recently surprised to discover a prevalent and nonpathogenic pig virus in GlaxoSmithKline's rotavirus vaccine Rotarix — a finding that raised questions about the safety of the vaccine while also highlighting sequencing's potential as a quality-control tool in vaccine development. Although not yet a common quality control tool, some biologics safety testing companies such as BioReliance, are already offering that service to pharmaceutical and biotech companies.
Prior to publishing their findings this week in the Journal of Virology, the researchers alerted the Food and Drug Administration, which recommended that physicians temporarily suspend use of the vaccine in the United States, although it added that there was no evidence suggesting that the vaccine posed a health safety risk.
"It's really surprising," said Jian Han, faculty investigator at the HudsonAlpha Institute for Biotechnology an expert in viral genomics and immunogenetics, who was not involved with the study. "I think it's an important finding that could have far-reaching influences in how we quality-control vaccines — [sequencing] technology can really help with that."
Eric Delwart, senior author of the paper and an investigator at the Blood Systems Research Institute in San Francisco, said he wanted to use sequencing technology to show that today's vaccines were clean and uncontaminated. "And, indeed, seven out of eight vaccines were free of eukaryotic viruses of concern," Delwart said. "We were quite surprised to find porcine circovirus 1 DNA in Rotarix."
The researchers used a metagenomics approach to analyze eight different live viral vaccine genomes, including vaccines for polio; rubella; measles; yellow fever; varicella-zoster (the virus that causes chickenpox); the measles, mumps, rubella combination vaccine; and two rotavirus vaccines.
First, they isolated the DNA, and then barcoded the DNA from each vaccine sample, amplified it, and then pool and sequenced it all in one run.
The team sequenced the vaccines in one lane of Roche's 454 GS FLX platform with Titanium chemistry, and generated over 500,000 reads with average read lengths of 311 base pairs, covering between 20 percent and 99 percent of the viral genomes.
The rotavirus vaccine Rotarix contained "6,344 reads with 98 percent identity to pig circovirus 1, covering the complete circular genome," the authors reported. The finding was confirmed with nested PCR.
Delwart added that the Rotarix vaccine has not demonstrated any known adverse health effects even though PCV1 has likely been present in the vaccine since it was originally produced. PCV1 has since been found in the original batch of cells from Rotarix's development, which he said is "reassuring, since all the clinical safety and efficacy tests that Rotarix passed with flying colors were made in the presence of PCV1."
GlaxoSmithKline spokesperson Sarah Alspach said preliminary investigations suggest that PCV1 may have come from trypsin, a protease that is frequently used in vaccine production for making the original cell bank, but that the company is continuing the investigation.
Aside from being an unexpected discovery, Delwart said the finding demonstrates sequencing's utility as a quality-control screen for vaccines.
"One thing is clear — this technology could be used not only for vaccines, but for other biologicals as well," Delwart added. "People can use these sequencing techniques early in development of vaccines to make sure they don't have these issues."
He said the cost should not be prohibitive, adding that the sequencing reagents for his study cost around $8,000 for all eight vaccines. He also said that shorter-read sequencing platforms should be just as effective as the 454 technology.
Current methods of screening vaccines for contaminants rely on PCR to look for specific viruses, so vaccine developers can't find a virus unless they are actively looking for it. "There are so many different types of virus, to do a PCR assay for each of them would be very expensive and take forever," Delwart said.
Alspach said that regulatory agencies are currently evaluating emerging technologies for their potential in vaccine production, and said that pending a decision, the company would work with the regulators.
"Where appropriate, GSK will work with regulatory agencies to adapt the regulatory standards and to integrate new molecular detection techniques in the testing of vaccines and the materials used in production," she told In Sequence in an email. "We can’t speculate on the potential use of viral metagenomics or any other emerging technology as an alternative quality control measure," she added.
Delwart added that pharmaceutical companies wouldn't have to do the sequencing themselves, but could employ the services of companies such as BioReliance, a biologics safety testing company, which uses microarrays and recently added 454-based sequencing in order to test client products for contaminants.
David Onions, chief scientific officer at BioReliance, said the company has been offering sequencing-based vaccine testing services to its clients for about a year. He said there are three main stages of vaccine production where sequencing has proven to be particularly helpful: the cell substrates, which are the cells used to produce the vaccine; the vaccine seeds, which are the viruses that are put into the cells and act as the master stock for the vaccine; and mammalian media, like serum, that is often used in vaccine production.
Onions said that those stages are "really where the quality control should be exercised, rather than the final product."
BioReliance recently published a paper in the journal Biologicals in which it demonstrated the use of transcriptome sequencing on the 454 platform to detect viruses in bovine serum. In the process, the company's researchers also found a previously unidentified bovine virus, the analysis of which they are preparing for publication.
Onions said that the company uses transcriptome sequencing because it can detect both the active, replicating, viruses and also any latent viruses. Whole-genome sequencing of the cell substrates doesn't provide any additional value, he added.
"Even if there are latent virus infections, they express some messenger RNA. There are latency-associated transcripts and also transcripts associated with tumor formation, and we can pick up those transcripts" with whole-transcriptome sequencing, he said.
Of particular concern, he said, are herpes virus and polyomavirus, which can be latent in cell lines and are also known to cause cancer.
Onions declined to reveal the pricing for BioReliance's sequencing services, but said that they are cost effective for the "characterization of key materials, like cell lines and virus seed, and also useful for some critical raw materials."
While he noted that sequencing is "probably not [suitable] as a standard quality-control measure for every virus at the moment" due to cost, he added that could change "soon."
HudsonAlpha's Han also said that the falling price of sequencing could make it a very cost-effective method for vaccine quality control.
"The cost [of sequencing] is coming down every day, and compared to safety issues and market consequences, this can really make a huge difference," he said.
Han added that sequencing could play an even larger role in the vaccine development process by helping to figure out why some people have different side effects and immune responses from a given vaccine. "You can use sequencing to follow the whole course of vaccines — evaluating their safety, efficiency, and differences in host response."