Archive for June, 2010

Dear New Scientist

6 June, 2010

In the full expectation that my letter will not see the light of day – nothing I have ever written to NS over some 15 years ever has – I will put this here, where more some people may see it.

Dear Editor:

I recall being a little miffed when I read the original article on
biodiversity in NS (24 April) – because there was no mention at
all of the greatest part of the biodiversity on this (and probably any
other) planet, which is viruses.  There are more viruses on Earth than
any other kinds of organisms, and virus genomes provide the greatest
source of gene diversity – yet they don’t rate a mention.

And then, in your Letters page of the 22 May issue, people take up
cudgels on behalf of fungi, of all things!

Cellism, that’s all it is….

Doctor, there’s a…pig virus in my vaccine??

6 June, 2010

rotavirus particle

I have for some time taught my third year students about how one must weigh relative risk vs. relative benefit when it comes to vaccination – with the Wyeth live rotavirus vaccine that was withdrawn in 2000 or so due to isolated incidents of intussusception (=telescoping of the bowel) as an object example.

Consider: the vaccine MAY have caused a couple of incidents (which granted, were serious) – but on the whole was protective, and well tolerated.  The publication referred to has this as the relative risk:

“…epidemiologic evidence supports a causal relationship, with a population attributable risk of ~1 per 10 000 (range of 1 in 5000 to 1 in 12 000) vaccine recipients.”

While this may be an unacceptable risk in a North American community – which is where it was tested, where children mostly just get sick from rotavirus infection – what about in a developing country, where the risk of an infant dying from rotavirus diarrhoea is far higher than 1 / 10 000?  Indeed, the same article says:

“Because perceptions of vaccine safety derive from the relative disease burdens of the illness prevented and adverse events induced, the acceptance of rare adverse events may vary substantially in different settings. [ my emphasis]”

Yes – like the vaccine may well have done a great deal of good, and very little harm, in a developing country setting where rehydration therapy is not the norm.  But it was pulled, and the world had to wait for Merck’s Rotateq pentavalent live vaccine and GSK’s Rotarix tetravalent live vaccine, YEARS later, and probably a lot of children died that may not have needed to.

I note that the Merck product has this as a warning, too:

“In post-marketing experience, intussusception (including death) and Kawasaki disease have been reported in infants who have received RotaTeq”.

So the vaccine has the same risk profile as Wyeth’s – yet it has been widely distributed, and is apparently highly effective – as is Rotarix.  In fact, in 2009 the WHO issued a recommendation “…that health authorities in all nations routinely vaccinate young children against rotavirus…”.

And then…news that must have made many a heart sink, in March 2010:

Pig virus contamination halts GSK Rotarix use

“GlaxoSmithKline’s oral rotavirus vaccine Rotarix has been temporarily shelved in the U.S. due to a pig-virus contamination. Researchers stumbled on DNA from porcine circovirus type 1–believed nonthreatening to humans–while using new molecular detection techniques. More work is being done to determine whether the whole virus or just DNA pieces are present.

Additional testing has confirmed presence of the matter in the cell bank and seed from which the vaccine is derived, in addition to the vaccine itself. So the vaccine has been contaminated since its early stages of development.”

The finding of the porcine circovirus type 1 (PCV-1) DNA in the vaccine was due to what seems to have been publication of an academic investigation in February 2010 of “Viral Nucleic Acids in Live-Attenuated Vaccines” by Eri L Delwart and team, mainly from Blood Systems Research Institute and University of California, San Francisco.  They used deep sequencing and microbial array technology to:

“…examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides.”

And they found:

“Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles [!!], while simian retrovirus (SRV) was present as genetically defective DNA.”

Whooooo…possibly live animal retroviruses in human vaccines??  But most importantly for our purposes:

Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids.”

I don’t know about you, but I’d be more worried about the retroviruses!  The authors concluded [my emphases in bold and red]:

“Given that live-attenuated viral vaccines are safe, effective, and relatively inexpensive, their use against human and animal pathogens should be encouraged. The application of high-throughput sequencing and microarrays provides effective means to interrogate current and future vaccines for genetic variants of the attenuated viruses and the presence of adventitious viruses. The wide range of sequences detectable by these methods (endogenous retroviruses, bacterial and other nucleic acids whose taxonomic origin cannot be determined, and adventitious viruses, such as PCV1) is an expected outcome of closer scrutiny to the nucleic acids present in vaccines and not necessarily a reflection of unsafe products. In view of the demonstrated benefit and safety of Rotarix, the >implications (if any) for current immunization policies of the detection of PCV1 DNA of unknown infectivity for humans need to be carefully considered.”

So they find all these bits of adventitious nucleic acids in a live human vaccine, then tell us it’s all right?  They go to say, however:

“As an added note, recent testing by GSK indicates that PCV1 was also present in the working cell bank and viral seed used for the generation of Rotarix used in the extensive clinical trials that demonstrated the safety and efficacy of this vaccine. These trials indicate a lack of detectable pathogenic effects from PCV1 DNA on vaccinees.”

So: a clinical trial in retrospect, then??  Interesting idea, that – it’s OK because they inadvertently tested it already and no obvious harm came of it!  Mind you, the same thing happened with SV40 in poliovirus vaccines over a lot longer period and on a much larger scale – and while the jury is still out on long-term effects, it appears as though there were none.

The first outcome of the finding, though, was that the FDA recommended in March that use of Rotarix be suspended, pending further investigations.

The same GSK press release reminds us that:

“Rotavirus is the leading cause of severe gastroenteritis among children below five years of age and it is estimated that more than half a million children die of rotavirus gastroenteritis each year – a child a minute [my bold – Ed]. It is predicted that rotavirus vaccination could prevent more than 2 million rotavirus deaths over the next decade. The continued availability of rotavirus vaccines around the world remains critical from a public health perspective to protect children from rotavirus disease. “

Cementing the risk/benefit argument very firmly and pre-emptively, then!

The next development was that Merck’s Rotateq, initially thought to be free of PCVs, was found to contain both PCV-1 AND PCV-2 DNA.  From their press release:

“In March 2010, an independent research team and the FDA tested for PCV DNA in rotavirus vaccines; at that time, PCV DNA was not detected in ROTATEQ by the assays that were used initially. Subsequently, Merck initiated PCV testing of ROTATEQ using highly sensitive assays. Merck’s testing detected low levels of DNA from PCV1 and PCV2 in ROTATEQ. Merck immediately shared these results with the FDA and other regulatory agencies.”

Alarming at first sight – but a variety of someones had done their relative risk calculations, and by mid-May, both vaccines had been cleared by the FDA – much to Merck and GSK stockholder relief, one imagines.

“The agency’s decision follows a May 7 recommendation from an FDA advisory panel, which said the PCV contamination didn’t appear to be harmful to humans and the vaccines’ benefits outweighed any “theoretical” risk the products might pose.

In announcing its decision, FDA said that both vaccines have strong safety records, including clinical trials of the vaccines in tens of thousands of patients, plus clinical experience with their administration in millions more. PCV isn’t known to cause illness in humans, whereas the rotavirus these vaccines ward off can cause severe illness and even death.”

All in all, what appears to be a sensible, logical decision, based on evidence – whether collected in retrospect or not – and common sense.  After all, as GSK points out in a press release,

“[PCV] is found in everyday meat products and is frequently eaten with no resulting disease or illness.”

Like plant viruses in vegetables, retroviruses in undercooked chicken and other meat, and a myriad other viruses and bacteria that live in, on, and with us – you really can’t keep away from everything.

But there’s still a good case to be made for killed vaccines….

Venter can do WHAT for influenza??

5 June, 2010

I have kept out of commenting on what J Craig Venter and others have done recently, given that many others have done so, and done so well – however, there is recurring mention of what “this technology” could do for influenza vaccines specifically, which has both puzzled and intrigued me, given a distinct lack of obviousness.

So I will comment, if only to clarify this issue for me and anyone else who cares.

To give some background, the New Scientist issue of the 29th of May has a guest editorial by J Craig Venter, Clyde Hutchison and Hamilton Smith, where they discuss some of the implications of their having made a totally synthetic and viable Mycoplasma mycoides genome (see also Science, DOI: 10.1126/science.1190719).

So what, exactly, is it they did?  OK, so they spent US$40 million or so constructing a genome, in segments, from sequence information housed in an electronic database, via chemical synthesis of long stretches of DNA.  They then assembled these segments into a singular genome in yeast, and then inserted this into cells of the closely-related Mycoplasma capricolum which had been stripped of their genomes – and incidentally, rendered unable to destroy the incoming genome as “foreign”, by a process which is now proprietary.  These cells then expressed the new DNA, which allowed them to multiply, and to take on all of the characteristics of the synthetic M mycoides, given that all of the original cell constituents from the original bug (proteins, mRNA, membranes, etc) would be turned over in time, and become those specified by the new genomes.

This is a big deal – a really, really big deal – but at the same time, they themselves recognise it is an incremental step in a long series of steps that started with Arthur Kornberg’s lab making the first complete synthetic and viable genome of a virus (phiX174) by in vitro synthesis from virion DNA, polymerase and nucleotides.  In fact they modestly point out that this is not even the first  complete cell genome that has been synthesised; it is the largest, however, and the one that worked.  They were not so modest in missing out a few other landmarks before their own complete synthesis of phiX174 in 2003, however: for example, the first synthesis of a functional plasmid, and the first generation of a RNA virus genome from a cDNA copy, and the complete synthesis of an infectious poliovirus genome, are not mentioned.

So what is it they did not do?

Well, they did not “create life”, however much even relatively respectable publications might claim they did: life is a lot more complex than chemicals, and people have “rebooted” cells before with exogenous genomes; what they did is not really qualitatively different to infecting a cell with a synthetic virus.

They have also not done anything that is immediately useful: their new organism differs from the original only in having a few genes missing, and a long literary message and ownership-encoding “watermark” inserted.

More positively, they have also most emphatically not opened the floodgates for bioterrorists to mail order complete poxvirus or anthrax genomes: as I have noted here previously,

“…There are more than enough nasty agents out there that are relatively easy to obtain, and do simple kitchen-based microbiology with, to keep entire cave complexes and Montana libertarian enclaves busy for years, without resorting to complicated molecular biology”.

Or spending $40 million dollars.  And I will say it again….

So aside from the details, what have they done?  In the NS editorial, Venter et al. say this:

“We now have the means to design and build a cell that will define the minimal set of instructions necessary for life, and to begin the design of cells with commercial potential, such as fuel production from carbon dioxide. We can assemble genome-sized stretches of DNA that can also be used to mix and match natural and synthetic pieces to make genomes with new capabilities.

Synthesising DNA in this way is still expensive, but we expect the cost to fall dramatically. This may make the complete synthesis of genomes competitive with the alteration of natural genomes to add new capabilities to bacterial cells. It should also be practical to synthesise simple eukaryotes, such as yeast, to which it is already possible to add extra chromosomes. The construction of large pieces of synthetic DNA and their introduction into a receptive cytoplasm is no longer a barrier. The limits to progress in synthetic biology are now set by our ability to design genomes with particular properties.”

Right: so what they have done is set the benchmark for what is possible – rather than what should be done.

Because it is a lot easier to do things such as they propose by other ways – as is pointed out in the companion article to the NS editorial.  For that matter, I am sure one could more easily end up with a completely synthetic and much larger cellular genome by incrementally replacing genome chunks by homologous recombination or transposon-mediated insertion / Cre-Lox deletion, and have it cost far less and be less subject to error, than by synthesising it de novo.

Influenza virus - Copyright Russell Kightley Media

And how does any of this relate to influenza vaccines?

The only comment I can find in the NS article that sheds light on this is the comment:

“As soon as next year, the flu vaccine you get could be made synthetically,” he [Venter] says.

Except that this has been possible for years already, after the poliovirus synthesis…?  I think it was rather a badly-chosen example rather than any actual plan; however, there is not a lot of point in making a synthetic influenza virus genome, given that the attenuated originals already work quite well as vaccines – and we don’t yet understand how to specify avirulence in influenza, so any synthetic version would necessarily be a copy of an extant version.

So hype rather than fact for ‘flu; promise rather than substance for carbon dioxide sequestration and biofuels – but still the coolest thing since sequencing your own dog…B-)