FDA clears rotavirus vaccines

The US Food and Drug Administration has updated its recommendations on both Rotarix and RotaTeq, vaccines for the prevention of rotavirus disease in infants:

Based on careful evaluation of a variety of scientific information, FDA has determined it is appropriate for clinicians and health care professionals to resume the use of Rotarix and to continue the use of RotaTeq.

In making its recommendation, the FDA considered the strong safety records of both vaccines, including clinical trials in tens of thousands of individuals and the use of the vaccines in millions of recipients. There is no evidence that either porcine circovirus type 1 or type 2 poses a safety risk to humans, and neither virus is known to infect humans or cause disease.

The FDA also noted that the benefits of the rotavirus vaccines are considerable and outweigh the small theoretical risk of the viral contaminant.

The product labels will be updated to reflect the fact that the vaccine contains a PCV contaminant. In addition, GlaxoSmithKline will rederive Rotarix so that it does not contain PCV. Merck has not yet made a decision about whether they should produce a PCV-free rotavirus vaccine. But if my suggestion carries any weight at Merck (I know it does not), they should not hesitate to follow GlaxoSmithKline’s lead.

Porcine circovirus DNA found in RotaTeq

The US Food and Drug Administration recently recommended that administration of Glaxo SmithKline’s Rotarix vaccine, which protects against rotavirus infection, be suspended after an independent research group found that the vaccine contains DNA of porcine circovirus type 1. Now the FDA reports finding circovirus DNA in the rotavirus vaccine made by Merck:

FDA recently received information from Merck & Co, Inc. that its preliminary studies have identified fragments of DNA from PCV1 and from a related porcine circovirus type 2 (PCV2) in its RotaTeq vaccine. Merck’s findings suggest that the number of PCV DNA fragments in its vaccine may be smaller than what has been found in Rotarix.

FDA has so far not recommended suspension of RotaTeq use. The Vaccines and Related Biological Products Advisory Committee meets today, after which FDA will make further recommendations on the use of the licensed rotavirus vaccines in the United States.

The source of porcine circovirus type 1 DNA in both Rotarix and RotaTeq is likely to be trypsin used during propagation of the cell cultures necessary for vaccine production. The enzyme is produced from porcine pancreas.

Update 10 May 2010: The FDA has posted a briefing document for the VRBPAC meeting that was held this past Friday. The Los Angeles Times reports on the outcome of the meeting:

Members of a Food and Drug Administration advisory committee meeting Friday urged physicians to continue using both commercial rotavirus vaccines despite evidence that both carry trace contaminants from a harmless pig virus. The panel did not take a formal vote on a recommendation, but a majority of participants said they thought the benefits of the vaccine far outweigh any potential risk. An agency spokeswoman said it would make no immediate recommendation on the vaccines, but would act expeditiously to come to a decision.

Finally, there are some good quotations from committee members in the article at Medscape Today.

TWiV 77: Non-nuclear proliferation

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich revisit circovirus contamination of Rotarix, then discuss poxvirus-like replication of mimivirus in the cell cytoplasm, and whether seasonal influenza immunization increases the risk of infection with the 2009 H1N1 pandemic virus.

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Do you want to know what is in your vaccines?

The recent discovery of contaminating porcine circovirus 1 DNA in Rotarix underscores the power of deep sequencing to ensure the purity of viral vaccines. The price of deep sequencing is now low enough that it is possible to use this technology to examine not just viral vaccines, but any biological product produced in mammalian cells for the presence of adventitious agents.

Some individuals have told me they do not want to know what is in their vaccines. Their logic is that even if we do identify a contaminating virus, we might not know if it is of any consequence to human health. But we would nonetheless worry about its presence and unnecessarily spend millions of dollars to remove it. The contrary view is that, although infection with porcine circovirus 1 might be benign, who can be confident that in 20 years the infection will not lead to dire consequences?

I am interested in what readers of virology blog think about this issue. Let your view be known by taking the poll below.

Deep sequencing reveals viral vaccine contaminants

Circovirus_genomeUse of the rotavirus vaccine Rotarix in the US has been temporarily suspended because the vaccine was found to be contaminated with porcine circovirus 1 DNA. The discovery was made in the laboratory of Dr. Eric Delwart, who has provided insight into what was found in Rotarix, and why he was analyzing the vaccine.

Delwart and his colleagues examined the purity of a number of human vaccines:

A little historical perspective about why we decided to analyze these vaccines even though there was absolutely no indications whatsoever that they are at all unsafe. Our intent was to use the latest technologies to show that live attenuated vaccine only contained the expected viral genomes and no other.

The Delwart laboratory obtained samples of eight infectious attenuated viral vaccines from the manufacturers: oral poliovirus vaccine (OPV, Bharat Biotech), rubella (Meruvax-II, Merck), measles (Attenuvax, Merck), yellow fever (YF-Vax, Sanofi Pasteur), human herpes 3 (Varivax, Merck), rotavirus (Rotarix, GlaxoSmithKline; and Rotateq, Merck) and multivalent measles-mumps-rubella (MMR-II, Merck). The vaccines were treated with DNAse and RNAse to remove nucleic acids that are not protected by viral capsids. Nucleic acid was then extracted from the vaccine, amplified by polymerase chain reaction, and subjected to DNA sequencing. A total of 501,753 sequence ‘reads’ were done.

The sequence analysis revealed the expected vaccine strains in each preparation, and in three cases, other unexpected viral sequences. The retrovirus avian leukosis virus was found in the measles vaccine, but at a very low level (700 nucleotides from 4 sequence reads). A virus similar to simian retrovirus was identified in Rotateq (276 nucleotides from 1 sequence read). Significant levels of porcine cirovirus 1 were found in Rotarix. The entire viral genome sequence was deduced from 6344 sequence reads, comprising over 40% of the reads done for that vaccine.

The avian leukosis virus sequences found in the measles vaccine are in intact virions, as DNA treatment of the vaccines did not prevent their detection by polymerase chain reaction (PCR). However simian retrovirus DNA was not detected by PCR after DNAse treatment of Rotateq. Therefore the viral DNA detected in Rotateq vaccine most likely originates from host DNA present in the vaccine preparation. The cells used to produce Rotateq are Vero cells – African green monkey kidney cells. A defective form of simian retrovirus DNA, called proviral DNA, is integrated into Vero cell DNA.

How did a porcine virus contaminate Rotarix, which is produced in Vero cells? The answer is not known, but the authors speculate that the culprit might be porcine trypsin, which is used during the propagation of Vero cells. Over 100,000 porcine circovirus 1 DNA molecules were detected in each vaccine dose, fully 10 times higher than the amount of rotavirus present. However, it’s not known if the porcine circovirus present is infectious.

There is little evidence that the presence of porcine circovirus 1 DNA in Rotarix constitutes a danger. Because both porcine circovirus 1 and 2 are common in healthy pigs, it’s likely that humans are frequently exposed to these viruses by eating pork or when workers at swine farms inhale pig feces. Both viruses are often found in human stool. Whether or not these viruses can replicate in humans is currently a matter of debate.

According to the FDA, “In four to six weeks, FDA will convene an expert advisory committee and make additional recommendations on the use of rotavirus vaccines.” What do they mean by this statement? According to Delwart,

I believe that GSK and FDA are now testing whether PCV1 in Rotarix can replicate in human cells and/or determine if vaccinated children are sero-positive to PCV1. If those sorts of tests are negative it will indicate that PCV1 is most likely not infectious for humans and therefore not a health concern in Rotarix.

Delwart believes that his work should not cast a negative light on viral vaccines:

For now it is my hope that people will not interpret this study as putting vaccines in a bad light but rather as reflecting the intense attention placed on vaccine safety using all the latest tools. It is only because of cheaper sequencing that these sorts of studies are possible. People should not be alarmed or over-react to every virus detection unless there is concrete evidence of human infection and pathogenicity. Viral metagenomics can both discover new viruses and improve the safety of existing vaccines. Its use early in the development of vaccines should help ensure their purity.

Victoria, J., Wang, C., Jones, M., Jaing, C., McLoughlin, K., Gardner, S., & Delwart, E. (2010). Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. Journal of Virology DOI: 10.1128/JVI.02690-09

TWiV 75: Rabies rampant

Hosts: Vincent Racaniello, Alan Dove, and Matthew Frieman

Vincent, Alan, and Matt review contamination of Rotarix with circovirus DNA, antigenic similarity between 1918 and 2009 H1N1 influenza, a collection of rabies reports, and chicken pox mistaken for smallpox in Uganda.

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Is circovirus DNA infectious?

Circovirus genomeThe US Food and Drug Administration does not want Rotarix, the rotavirus vaccine, to be used because it contains porcine circovirus 1 DNA. If complete copies of the circovirus genome were present, would they constitute a potential threat to recipients? Put another way, is circovirus DNA infectious?

Here is the information you need to answer this question.

  • The circovirus genome is a circular, single stranded DNA molecule (pictured).
  • To infect a cell, the two viral proteins encoded in the DNA must be produced.
  • To produce proteins, mRNA must be synthesized from the viral DNA.
  • Single-stranded DNA cannot be copied  into mRNA; the DNA must be double-stranded.
  • The circovirus particle consists of a protein shell surrounding the viral DNA. There are no other components in the virion.
  • During infection of cells by circoviruses, the particles enter the nucleus where the viral DNA is released
  • If naked DNA is added to cells, a good fraction ends up in the nucleus.

Knowing these facts, can you determine whether introduction of circovirus DNA into cells would lead to viral replication?

Porcine circovirus DNA in rotavirus vaccine

Circovirus_genomeThe US Food and Drug Administration has recommended that administration of the Rotarix vaccine, which protects against rotavirus infection, be suspended. This action comes after an independent research group found that the vaccine contains DNA of porcine circovirus type 1.

Rotaviruses are the single leading cause of diarrhea in infants and young children. Each year rotavirus gastroenteritis causes over 1,250,000 episodes of diarrhea and 527,000 deaths, mainly in developing countries. Rotavirus vaccines are used to reduce the global burden of rotavirus disease. Rotarix, the vaccine manufactured by GlaxoSmithKline, is an infectious, attenuated vaccine that is administered orally to infants.

The genetic information of rotaviruses consists of 11 segments of double-stranded RNA. In contrast, porcine circoviruses are small viruses with a circular, single stranded DNA genome (pictured). At 1.7 kb in length, the DNA is among the smallest known viral genomes and encodes only two proteins. Porcine circovirus 1 was originally discovered as a contaminant of a pig kidney cell line. Later a second strain, porcine circovirus 2, was isolated and shown to be associated with postweaning multisystemic wasting disease, an emerging disease of swine.

Porcine circovirus 1 DNA was also found in the cells used to produce Rotarix. Therefore the contaminant has been present since the early days of vaccine development, including clinical trials. A different rotavirus vaccine produced by Merck, called RotaTeq, does not contain porcine circovirus DNA.

It’s not at all clear that the presence of porcine circovirus 1 DNA in Rotarix is a problem. Circoviruses have not been associated with human disease, and porcine circovirus 1 has not been found to cause disease in any animal. Many humans have antibodies to these viruses, including porcine circovirus, indicating that they were infected at one time. Furthermore, it’s not known if the vaccine contains infectious virus or DNA fragments. Nevertheless, it’s always preferable to err on the side of caution, as the FDA has done.

Because porcine circoviruses are widespread in commercial swine populations, there have been concerns about the use of porcine organs for xenotransplantation and for the production of products used in humans such as factor VIII, heparin, insulin and pepsin. It is therefore important to ensure that such products do not contain infectious circoviruses.

This incident will undoubtedly further increase public distrust of vaccines and vaccine manufacturers. I have not yet seen the journal article describing these findings, so I can’t comment on why the contaminant was not identified early in vaccine development. According to the FDA, “In four to six weeks, FDA will convene an expert advisory committee and make additional recommendations on the use of rotavirus vaccines.” If Rotarix is found to contain infectious porcine circovirus, then its use will certainly be discontinued. However, detection of small noninfectious fragments of porcine circovirus DNA Rotarix will likely lead to resumption of vaccine use. In either case, new lots of vaccine should be produced using circovirus-free cells.

Tischer I, Bode L, Apodaca J, Timm H, Peters D, Rasch R, Pociuli S, & Gerike E (1995). Presence of antibodies reacting with porcine circovirus in sera of humans, mice, and cattle. Archives of virology, 140 (8), 1427-39 PMID: 7544971

Fenaux M, Opriessnig T, Halbur PG, Xu Y, Potts B, & Meng XJ (2004). Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product. The Journal of general virology, 85 (Pt 11), 3377-82 PMID: 15483254