February 2005 Archives

Just as I was banishing my ignorance about how the forthcoming trial vaccine for H5N1 was produced, a trio of excellent articles from the Wall Street Journal and Fortune landed in my Inbox, facilitating my education.  The short story is that the virus grown in chicken eggs as the source of an attenuated vaccine is not actually H5N1.  The genes that cause the virus to be so fatal to eggs have been replaced with genes from less virulent strains, while the HA protein on the surface of the virus is modified so that it is more stable.

Alas, because the WSJ doesn't allow you to look at their list of stories in the print edition without a subscription, I can't even provide links to the stories.  Fortune, evidently, is more forthcoming.  Here are the titles, etc;

"A primer on the Threat of Avian Flu...", by Gautam Naik, and "Avian Flu Poses Challenge to Global Vaccine Industry...", by David Hamilton and Gautam Naik; both are from the 28 Feb, 2005 issue of the WSJ.  "The Coming War Against Bird Flu", by David Stipp, will appear in the 7 March, 2005 issue of Fortune.

The upshot of the three articles is that the vaccine is produced in sterile chicken eggs via a recombinant virus that is a modified version of H5N1.  This strategy requires a large number of those eggs, which are not easy to come by, and produces a vaccine that prompts the production of antibodies against a virus that may, or may not, be similar to the wild type H5N1.  That is what human trials will have to determine.

Thus my initial concerns (here and here) about this issue were not so far off target.  Stipp's article does an excellent job describing the production of the vaccine, and associated challenges.  It is pretty clear we need to come up with alternative means of producing vaccines, preferably rapid synthetic approaches that are deployable from a distributed infrastructure.

UPDATE (7 March 2005): I stumbled over this article in The Scientist, "H5N1 vaccine strain in a week", from 29 January 2004, which opens;

A prototype vaccine strain of the H5N1 flu virus causing havoc in Asia will probably be ready next week, John Wood of the UK National Institute for Biological Standards and Control (NIBSC) told The Scientist today (January 29). However, months of other hurdles remain before it may be ready for public health use.

The article describes several genetic manipulations of the H5N1 strain that will make it easier to produce in chicken eggs, beginning with the removal of, "a stretch of 4 or 5 basic amino acids at the hemagglutinin cleavage site that allows the virus to replicate in every organ of a chicken's body, rather than respiratory and gut tissue normally infected".

The article cites Klaus Stohr as saying, "The H5N1 virus kills chicken eggs, the normal medium for growing flu vaccine viruses, so the WHO laboratories are using reverse genetics to lower the pathogenicity of the virus to chickens and to get a high yield in the egg cultures", and describes the additional genetic manipulations; "Using other lab strain flu plasmids containing the other components of the viral genome, the team will then reassort the pieces into a nonpathogenic vaccine strain." 

Finally, the article suggests that, "Sufficient amounts of safety-tested prototype vaccine virus will probably be available for the necessary 1 to 2 months of clinical trials in the next 4 weeks".  The date of this article, again, was 29 January, 2004.

Looks like we are well on our way, circa January 2004, to producing a lovely vaccine against a bug that doesn't actually exist in nature.  We clearly need an alternative to attenuated (or killed) whole virus vaccines.  When I have time, I will post what I have been learning about DNA vaccines.

"Bird fly outbreaks may go unnoticed in humans", a news piece in the 26 February, 2005 issue of New Scientist, reports that human cases of Avian Flu (H5N1) may be misdiagnosed.  Several patients in SE Asia have presented with symptoms unusual for the flu, and only after death did they test positive for the virus.  The piece also reports that the WHO is "analysing blood samples from people in areas affected by h5N1 to see how many carry antibodies against the virus".

The difficulty here is that it can take up to several weeks (say, one to three, depending on the etiology of the bug) for the adaptive immune response to produce antibodies against a pathogen.  It appears that people are dying within that time frame, which means that testing for antibodies is unlikely to be a useful diagnostic tool, at least given standard assay sensitivities.  Using reverse-transcriptase PCR (RT-PCR), it may be possible to detect the RNA genome of the bug, but clinical PCR is a true art.  It is often quite difficult to see anything via PCR in a clinical sample, unless you can really clean it up via purification.  That purification, however, particularly in the case of RNA, tends to reduce the sensitivity of the assay by removing or destroying the target nucleic acids before the amplification step.

I still haven't been able to determine what magical means will be used to produce a vaccine against the H5N1 strain of Avian Flu.  Press is very thin on how production and testing of the vaccine is going.  Yet policy decisions are being made based on the notion that the vaccine will be available in quantity soon.

A press release on the CIDRAP site from the World Health Organization notes that WHO will probably recommend governments start stockpiling vaccines against H5N1.  The release also cites unnamed "U.S. officials" who say that clinical trials of vaccines from Chiron and Sanofi-Pasteur are supposed to start soon, while also noting that, "H5N1 may not match the pandemic strain, the vaccine's shelf life of up to 2 years is relatively short, and, because companies have not yet begun clinical trials, licensing of the vaccine is months away."

And in another release, the CIDRAP site quotes Michael Osterholm, who is director of the University of Minnesota Center for Infectious Disease Research and Policy, "We don't have a pandemic strain of vaccine yet, and we don't have any idea whether any of the vaccines to date would be efficacious."  In the Technological Challenges to Vaccine Development section of the Pandemic Influenza overview at CIDRAP, we find; "Highly pathogenic avian strains cannot be grown in large quantities in eggs because they are lethal to chick embryos."

To the extent that we should trust the popular press on this issue, as part of a short story on What You Need to Know About Avian Flu, the 9 February, 2004 issue of Business Week states, "Vaccines are usually produced in chicken eggs, but H5N1 is lethal to fertilized eggs."

Yet a 24 February, 2005 story on Newsday.com says, "Two million doses of vaccine are being stored in bulk form for possible emergency use and to test whether it maintains its potency," while 8000 doses are, "nearly ready to be shipped to the National Institute for Allergy and Infectious Diseases for clinical trials."

Perhaps the vaccine about to enter trials is from source other than chicken eggs?

In 10 February, 2005 testimony before the The Committee on Government Reform, Jesse L. Goodman, Director of the Center for Biologics Evaluation and Research at the FDA, while describing how the Department of Health and Human Services will spend roughly a billion dollars over the next few years on influenza related activities, said; "While work remains to obtain sufficient vaccine yields and evaluate cell-based vaccines for their safety and effectiveness, moving from an egg-based production to a cell-culture production can potentially shorten the time needed to produce vaccine as well as decrease the risk of contamination inherent in egg-based production."  That is, there isn't yet a functional alternative to using chicken eggs to produce vaccine.

So what gives?  I can only speculate that details about the vaccine are being closely held until more is learned about how it behaves in humans.  But with so many sources suggesting the vaccine can't be grown in eggs, I have to wonder what tricks Chiron and Sanofi-Pasteur have come up with to produce it in bulk.  Perhaps it is a low yield process and they have concentrated the virus produced from a much larger number of eggs?

I wish someone would come out and clearly explain where the vaccine is coming from and how it is produced.  The issues of what infrastructure exists to make vaccines, how much can be made, and whether it will be effective are quite critical for charting our course as we prepare for a potential pandemic.

Here is part of "The Second Coming of Synthetic Biology", the fifth chapter of my book, Learning to Fly: the past, present, and future of Biological Technology.  More at: www.BiologyIsTechnology.com.

Chapter 5.  The Second Coming of Synthetic Biology

"I must tell you that I can prepare urea without requiring a kidney of an animal, either man or dog.”   With these words, in 1828 Friedrich Wohler announced he had irreversibly changed the world.  In a letter to his former teacher Joens Jacob Berzelius, Wohler wrote that he had witnessed, “The great tragedy of science, the slaying of a beautiful hypothesis by an ugly fact.”  The beautiful idea to which he referred was vitalism, the notion that organic matter, exemplified in this case by urea, was animated and created by a vital force and that it could not be synthesized from inorganic components.  The ugly fact was a dish of urea crystals on his laboratory bench, produced by heating inorganic salts.  Thus was born the field of synthetic organic chemistry.

Around the dawn of the 19th century, chemistry was in revolution right along with the rest of the western world.  The study of chemical transformation, then still known as alchemy, was undergoing systematic quantification.  Rather than rely on vague and mysterious incantations, scientists such as Antoine Lavoisier wanted to create what historian of science and technology Bruce Hevly calls an “objective vocabulary” for chemistry.  Through careful measurement, a set of clear rules governing the synthesis of inorganic, non-living materials gradually emerged.

In contrast, in the early 1800s the study of organic molecules was primarily concerned with understanding how molecules already in existence were put together.  It was a study of chemical compositions and reactions.  Unlike the broader field of chemistry taking shape from alchemy, making new organic things was of lesser concern because it was thought by many that organic molecules were beyond synthesis.  Then, in 1828, Wohler synthesized urea.  Suddenly, with one experiment, the way scientists did organic chemistry changed.  The ability to assemble organic molecules from inorganic components altered the way people viewed a large fraction of the natural world because they could conceive of building much of it from simpler pieces.  Building something from scratch, or modifying an existing system, requires understanding more details about the system than simply looking at it, poking it, and describing how it behaves.  This new approach to chemistry helped open the door to the world we live in today.  Products of synthetic organic chemistry dominate our environment, and the design of those products is possible only because understanding the process of novel assembly revealed new principles.

It was this step of moving to Synthetic Chemistry, and then to an engineering of chemistry, which radically changed the way people understood chemistry.  Chemists had to learn rules that weren’t apparent before.  In the same way that Chemical Engineering changed our understanding of nature, as we begin engineering biological systems we will learn considerably more about the way biological pieces work together.  Challenges will arise that aren’t obvious just from watching things happen.  With time, we will understand and address those challenges, and our use of biology will change dramatically in the process.  The analogy at this point should be clear; we are well on our way to developing Synthetic Biology.

Before going further, it is worth noting that this is not the original incantation of the phrase “synthetic biology”.  Whatever the reception this time around, the first time it was a flop.  In her history of the modern science of biology, Making Sense of Life, Evelyn Fox Keller recounts efforts at the turn of the 20th Century to discover the secret of life through construction of artificial, and synthetic, living systems; “To many authors writing in the early part of the [20th] century, the [path] seemed obvious: the question of what life is was to be answered not by induction but by production, not be analysis but by synthesis.”(Keller, p.18)  This offshoot of experimental biology reached its pinnacle, or nadir, depending on your point of view, in attempts by Stephané Leduc to assemble purely physical and chemical systems that demonstrated behaviors reminiscent of biology.  As part of his program to demonstrate “the essential character of the living being”(ibid, p.28) at both the sub-cellular and cellular level, Leduc constructed chemical systems that he claimed displayed mitotic division, growth, development, and even cellular motility.  He described these patterns and forms in terms of the well-understood physical phenomena of diffusion and osmotic pressure.  It is important to note that these efforts to synthesize life-like forms relied as much on experiment as upon theory developed to describe the relevant physics and chemistry.  That is, this was a specific program to use physical principles to explain biological phenomena.  These efforts were described in a review paper at the time as “La Biologie synthetique”(ibid, p.31-32).

While the initial reception to this work was somewhat favorable, Leduc’s grandiose claims about the implications of his work, and a growing general appreciation for complicated biological mechanisms determined through experiments with living systems, led to something of a backlash against the approach of understanding biology through construction.  By 1913, one reviewer wrote, “The interpretations of M. Leduc are so fantastic…that it is impossible to take them seriously”(ibid, p.31).  Keller chronicles this episode within the broader historical debate over the role of construction and theory in biology.   History regards the folks in the synthetic camp, and related efforts to build mathematical descriptions of biology, particularly in the area of growth and development, as poorly regarded by their peers.  Perhaps inspired by the contemporaneous advances in physics, it seems that the mathematical biologists and the synthetic biologists of the day pushed the interpretation of their work further than was warrented by available data.

In response to what he viewed as theory run rampant, Charles Davenport suggested in 1934 that, “What we require at the present time is more measurement and less theory…There is an unfortunate confusion at the present time bewteen quantitative biology and bio-mathematics…Until quantitative measurement has provided us with more facts of biology, I prefer the former science to the latter”(ibid, p.86).  I think these remarks are still valid today.  Leduc, and the approach he espoused, failed because real biological parts are more complex, and obey different rules, than his simple chemical systems, however beautiful they were.  And it is quite clear that vast forests have been felled to publish theory papers that have little to do with the biology we see out the window.  But theory, drawn from physics, chemistry, and engineering, does have a role to play in describing biological systems.  Resistance to the tools of theory has been, in part, cultural.  There has always been a certain tension in biology over the utility of mathematical and physical approaches to the subject;

To put it simply, one could say that biologists do not accept the Kantian view of mathematics (or, rather, mathematization) as the measure of a true science; indeed, they have often actively and vociferously repudiated any such criterion.  Nor have practicing biologists shown much enthusiasm for the use of mathematics as a heuristic guide in their studies of biological problems.(Keller, p. 81)

Fortunately, this appears to be changing.  Mathematical approaches are flourishing in biology, particularly in the interpretation of large data sets produced by genomic and proteomic studies.  Physicists and engineers are making fundamental contributions to the quantitative understanding of how individual proteins work in their biological context.  But I think it is important to acknowledge that not all biologists think a synthetic, bottom up, approach will yield truths applicable to complex systems that have evolved over billions of years.  Such concerns are not without merit, because as the quotation from Charles Davenport suggests, biology has traditionally had more success when driven by good data rather than theory.  The challenge today is to build quantitatively predictive design tools based on the measured device physics of real biological parts, and to implement designs within organisms in ways that work in the real world...

More at: www.BiologyIsTechnology.com.

I was the guest for the Immortality Institute's online chat tonight.  The archive is here. I did this as a  bit of an experiment, because I wasn't sure how well communicating this way would work.  Turns out it is mostly okay, though the timing of questions and answers turns out to be challenging.

(First, here is the NIH Focus on the Flu site.  Decent general info there.)

Klaus Stohr is the chief of the World Health Organization's global influenza program.  He is worried that we are overdue for a flu pandemic.  In this profile in The Lancet, he is attributed with the observations that flu pandemics occur on average every 27 years, that the last one hit 37 years ago in 1968, and that between 2 and 7 million people could die in the next pandemic.

As a veterinarian and influenza specialist, Dr. Stohr obviously knows a lot more about flu bugs than do I.  However, his statistics may need a second look, particularly for incidents in the past 100 years.  Arnold Monto notes in his New England Journal of Medicine perspective "The Threat of an Avian Influenza Pandemic" (27 Jan 2005) that, "There have been three influenza pandemics during the past century -- in 1918, 1957, and 1968."  It is true that the average interval between these three events is just under 30 years.  I don't know how many data points Dr. Stohr is working with, but the width of the distribution, in this case, is hardly even computable for pandemics this century.  The interval between events is just as likely to be 40 years as it is 30 (not so comforting, I admit).  In any event, given the state of modern medicine, travel, and sanitation (and the variability in all those things across the globe) nobody should be drawing firm statistical conclusions from the three most recent data points.  The point of this is that because this bug is not behaving as expected, perhaps we should reevaluate our expectations.

How much do we really know about pandemic strains?  Perhaps a good place to start is examining how similar the three 20th century strains were.  Not very, I am beginning to think.  Although each were a novel type A virus of avian origin, Monto observes that, "In 1957 and 1968, the new viruses had components of previous human viruses as well as avian viruses...it was determined retrospectively that in both cases, there had been a reassortment of avian and human genes -- most likely the result of the coinfection of a host by two different viruses."  Monto then notes that the 1918 strain appears to have resulted from mutation in an avian strain (see my post, The Spanish Flu Story).  So, we are down to two pandemic strains, out of only three total, that arose through the historically low probability process of reassortment (see the end of my post, A Confluence of Concerns).  The numbers aren't looking good for deriving general principles about potential pandemic flu strains.

Adding to the confusion is the fact that, according to Monto, "The genetic characteristics of [H5N1] are still completely avian; neither mutation nor the sharing of genetic material with a human virus has taken place."  (I don't entirely understand this statement in light of assertions that H5N1 is becoming more pathogenic in poultry -- how else would this occur than by mutation?  Or is recombination amongst avian strains the assumed mode of increase pathogenesis?)  Klaus Stohr himself, in a 27 Feb, 2005 editorial in NEJM, "Avian Influenza and Pandemics -- Research Needs and Opportunities", wonders;

Why has H5N1 not reassorted with a human influenzavirus?  It certainly has had ample opportunity to do so...Unprotected workers [destroying infected poultry have] had intense exposure, as did health care workers.  Virologic surveillance has demonstrated the concurrent circulation of human viruses.  Hence, one conclusion is tempting: if H5N1 could reassort, it should have done so by now.  The explanation may lie in sheer statistical luck.

Hmmm.  That's not so satisfying. 

The last thing I want to do here is undermine the efforts of experts to understand what is going on and to try to prevent a pandemic.  However, I can't square public statements about the risk we face with what data I find in the literature.  There is definitely a troublesome lack of information about how flu bugs work, how the evolve, and what we might do to stop them, particularly with vaccines.  This press release, dated 27 May 2004, from the National Institutes of Allergy and Infectious Disease, says vaccines against H5N1 will be made by Chiron and Aventis Pasteur using the traditional chicken egg method.  While I have informally heard that H5N1 is so lethal that it kills chicken embryos before they can produce an adequate amount of virus to use as a vaccine, I still haven't been able to confirm whether or not it is technologically possible to produce an H5N1 vaccine this way.

So what do we do?  Stohr, again; "Substantial gaps in knowledge remain, making the ability of science to guide policy imperfect at a critical time."

Indeed.

The first draft chapter of my book, tentatively titled Learning to Fly: the past, present, and future of Biological Technology, is is now up on the web at www.BiologyIsTechnology.com, along with the first few pages of some additional chapters.

I am stuck trying to post on a decrepit WebTV system from a hotel in Kauai.  Don't buy one of these things if it is your last option to communicate with the world.  Forget trying to include links in a post.  Regarding Oliver Morton's recent op-ed in the Times, "Biology's New Forbiden Fruit" (11 Feb, 2005), there was an editing mistake that gave me sole credit for the recent paper on the use of Tadpoles for sensitive detection in Nature Methods.  It seems this misprint will be corrected in a forthcoming issue.  Why oh why did I leave my Powerbook at home?

UPDATE (18.02.05, back in Seattle.  Brrr.):  Here is the column (NYTimes in exchange for your first born and all that), and here is a free copy at freerepublic.com, a site that I wouldn't ordinarily advertise, but they chose to violate copyright and take on the Times, which means I didn't have to.

UPDATE: Here is the correction in the Times, with an additional very odd addendum that seems to overplay the cautionary aspects of Oliver's op-ed.  Editors -- can't live with 'em, and can't live without -- hmmm...

The Center for Infectious Disease Research And Policy (CIDRAP), at the University of Minnesota, has an excellent web site for those interested in H5N1 and Pandemic Flu strains.  The site also covers Biosecurity, Bioterrorism, Food Safety, BSE, and SARS, amongst others, though I have yet to peruse those topics.

The section on "Pandemic Influenza" briefly mentions the problem that vaccines for H5N1 can't be made via the usual high-technology route of infecting chicken eggs because the virus kills the chick embryos too quickly.  I've heard this before, but can't find any references in my stash of PDFs.  Does anyone know of a decent paper/website/NY Times story that explains this in some detail?  Similarly for a review of efforts to grow virus in mammalian cell culture?

It's really quite embarrassing that we are stuck using century-old technology to combat these viruses.

In the 23 December 2004 issue of Nature, Jingdong Tian et al. describe a new method for "Accurate multiplex gene synthesis from programmable DNA microchips."  The name most frequently associated with the paper is that of George Church, a professor at Harvard Medical School.

The authors combine microfluidics, biochemistry, and molecular biology to produce a widget capable of rapid synthesis of long oligonucleotides (oligos).  The paper reports an integration of 1) a new way to elute completed oligos from arrays; 2) on chip amplification of oligos; 3) error correction using via "strict hybridization" conditions to remove mistakes; and 4) microfluidic multiplexing, to produce 14.5 kilobase (KB) long fragments of DNA.  Slipped in at the end of the paper is the claim that they have already used this technology to successfully fabricate 95-382 KB oligos, assembling them into megabase (MB) length sequences.  Although it may receive less press, when the paper comes out describing the latter advance it will mark a significant milestone in the human ability to manipulate biological systems.  Organismal length sequences will be well within reach.

Now for the press coverage of the paper.  Mr. Wade, in the 12 January 2005 edition of The New York Times, describes it thus;

Researchers have made an unexpectedly sudden advance in synthesizing long molecules of DNA, bringing them closer to the goal of redesigning genes and programming cells to make pharmaceuticals.

But the success also puts within reach the manufacture of small genomes, such as those of viruses and perhaps certain bacteria. Some biologists fear that the technique might be used to make the genome of the smallpox virus, one of the few pathogens that cannot easily be collected from the wild.

With all respect to George Church and his colleagues, and without reducing the significance of their technical achievement, I have to say this actually isn't so much of a surprise.  It is true that I have been following this, and that I saw the chip on Erdogan Gulari's desk last winter.  In other words, I have had time to get over it.  But this sort of thing has been in the air for a while, and Drew Endy and I talked about something similar many years ago at tMSI.  I am certain we were not the first to do so.

More interesting is the reduction in cost per base of the synthesis, which Professor Gulari puts at about a penny a base for the long oligos.  This is news, and the cost falls completely off the curves I published in 2002.  The impact of the paper will only be felt when the technology becomes widely available, which is at least a couple of years out.  Unless I misunderstand the market and the state of the technology, the only people with access to synthesis at this scale and cost are the authors of the paper and their pals in academia.

With respect to suggestions that oligo synthesizers should be regulated, my views are well known at this point.  In the NY Times piece, Professor Church suggests registration of instruments could go a long way towards increasing security.  More information is, of course, better.  But we have too much experience forcing people "underground" when the things they want to pursue are restricted or made illegal.  I suspect we will be much better off encouraging an open community of people unafraid to talk about what they are up to in their garage.  Finally, even if instrument makers are willing to going along with registration, there will be a big hole in the registry due to the aftermarket, and I don't know how to enforce registration of homemade DNA synthesizers.  There are arguments that no one will want to build a synthesizer, or to play with what it enables, but I think the history of tinkering is a fairly decisive counterexample.  So the real question is, how do you stop people from playing?  I don't think you can.

As an advance in the technology, far more interesting to me is a paper by Peter Carr et al, from the Jacobson group at MIT, "Protein-mediated error correction for de novo DNA synthesis".  They use the DNA mismatch-binding protein MutS to identify mistakes, which are then removed from the synthesis pool.  One round of this procedure improves the error rate to ~1 in 4000 bases, which is a factor of three better than the Tian et al work discussed above.  A second round of error correction reduces the error rate to ~1 in 10 KB.  This rate is so low that a single round of synthesis and cloning should be sufficient to produce multi-gene cassettes suitable for use in complicated genetic circuits.  The combination of the protein-mediated correction and the Tian et al work would be impressive indeed.  Since George Church is thanked in the acknowledgments of the Carr paper, no doubt all the right people are considering the possibilities.