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FDA Evaluates Illumina GA, 454 for Vaccine QC; Looks to Implement Method for Polio Vaccine


By Monica Heger

Scientists at the US Food and Drug
Administration have begun to explore next-gen sequencing's potential for quality control in the manufacture of vaccines and have so far tested the ability of Roche's 454 GS FLX and the Illumina Genome Analyzer to identify pathogenic mutations in live attenuated polio vaccine.

In a study published earlier this month in the Proceedings of the National Academy of Sciences, the FDA scientists concluded that sequencing could be the method of choice for monitoring vaccines and proposed to use the approach "for the sensitive detection and quantification of all mutations in the entire genome of attenuated vaccines."

"We believe this is an ultimate tool for monitoring the genetic stability of all viral vaccines — not only live, but also inactivated vaccines," Konstantin Chumakov, the associate director for research at the FDA's Office of Vaccines Research and Review, told In Sequence.

Chumakov added that his team is now working with the World Health Organization and other international regulatory agencies to see how the technique could be implemented as a quality-control measure for oral polio vaccine.

The live viral vaccine is no longer used in the US, but it is still used in other countries. One issue with the vaccine is that it can incur mutations that enable it to regain its neurovirulence. While all batches are tested using a PCR technique called mutant analysis by PCR and restriction enzyme cleavage, or MAPREC, the technique can only screen for mutations at a few known genomic loci. As a result, the vaccine must be tested in monkeys before it is deemed safe for use in humans. Chumakov said incorporating sequencing in the testing could negate the use of animal tests.

Additionally, Chumakov said that the FDA scientists plan to study the technique in other live viral vaccines such as the mumps, measles, yellow fever, West Nile, and influenza vaccines, although details of those projects have not yet been ironed out.

In the PNAS study, the researchers first sequenced one sample of oral polio vaccine that had passed the monkey neurovirulence test, and one sample that had not, using the 454 GS FLX. The sequencing was done at FDA contractor Science Applications International as part of the National Institutes of Health's advanced technology program. They achieved average read lengths of around 200 base pairs and sequenced each base an average of 10,000 times.

While the method clearly distinguished between the virulent and nonvirulent batches, it also predicted more insertions and deletions than the researchers expected. Suspecting that those mutations could be methodological artifacts rather than true mutations, the researchers next sequenced eight different batches of the viral stock using both the 454 and Illumina platforms.

The Illumina sequencing was done by Macrogen, with 38-base single-end reads, and yielded about 60 times more sequence data than the 454 method. For this experiment, each base was only sequenced by 454 885 times and by Illumina around 56,000 times. Each method detected about the same number of substitutions, but the 454 method falsely predicted more insertions and deletions. The false calls are a known issue, intrinsic to the pyrosequencing method, and because of that and the fact that Illumina sequencing produces so much more information, Chumakov said that for now, he prefers the Illumina method for vaccine sequencing. Improvements in the Illumina technology since the time of the study, such as paired-end sequencing and read lengths of around 100 base pairs, also tip the scales in Illumina's favor, he said.

Chumakov said that currently the FDA has no plans to invest in a sequencer, and would continue to contract out its sequencing. "It's quite an expensive technology and is evolving very fast," he said. "If we were to invest in one version of the technology, three years later it may be obsolete."

He added, however, that in the future, that decision could change. "As the technology stabilizes, there will be clear winners and we'll know which version of the technology is most suitable for us."

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A Surprise Finding

Earlier this year, Eric Delwart, an investigator at the Blood Systems Research Institute in San Francisco, unexpectedly discovered pieces of a porcine circovirus in GlaxoSmithKline's rotavirus vaccine using sequencing (IS 4/6/2010).

While that finding was published before the FDA's research, Chumakov said that the FDA had already begun to use next-generation sequencing to study the polio vaccine, and that the research was not spurred by that finding.

However, after Delwart's finding, the FDA's Vaccine's and Related Biological Products Advisory Committee met to discuss the implications of the results, and how next-gen sequencing could be incorporated into vaccine quality control (IS 5/11/2010).

The advisory committee, while making no formal recommendations to the FDA, concluded that next-gen sequencing technology represented a powerful tool for determining vaccine quality, but expressed concern over data interpretation and the logistics of implementing it into the regulatory process.

Now, though, Chumakov said that the FDA is interested in implementing the technology. Initially, sequencing would be especially applicable for testing live attenuated viral vaccines, although he said that eventually it would be appropriate for inactivated viral vaccines and possibly even bacterial vaccines.

Since presenting his results this summer, Chumakov said he's been approached by sequencing service providers and vaccine manufacturers interested in implementing sequencing into the vaccine production process. "It's really quite obvious that this is the way to go," he said.

He said that he expects to see vaccine submissions that include sequencing as a quality-control step within the next few years, though he acknowledged that incorporating sequencing into the regulatory process could be complicated. Because sequencing is such a non-biased approach, it turns up both known and unknown mutations.

"With all those thousands of mutations, we don't know what they all mean. Some may mean nothing," Chumakov said. "We're seeing things we haven't seen before."

For the oral polio vaccine, though, the mutations that are screened for confer virulence only when present in large quantities. Chumakov said that could also be the case for some of these unknown variants. "They may be a marker of consistency. If [the frequency of] one variant goes up, it may be a red flag that something has gone wrong."

Further work is needed to validate the technology and determine the acceptable range of variation. In the case of the oral polio vaccine, the FDA is already working with WHO, UNESCO, and other regulatory agencies about the best way to incorporate the information, and that could set the standard for how the technology is implemented into the production of other vaccines, Chumakov said.

Have topics you'd like to see covered in In Sequence? Contact the editor at mheger [at] genomeweb [dot] com.

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