(Not Quite Live From) Synthetic Biology 2.0, Part V :: Fin

First off, here is the link for Synthetic Biology 3.0, next year's meeting in Switzerland.

This year's meeting was impressive on many counts.  As I have noted already (Part II), there was a distinct change in the flavor of the presentations.  The first day started out with a Nobel Laureate, followed up by a potential (probable?) future Laureate.  There was a significant amount of money in the room, from corporate representatives of synthesis companies to venture capitalist Vinod Khosla.  With respect to the technical presentations, the sheer diversity of systems and applications compared to two years ago was remarkable.  People are playing with more organisms and more parts (here's the meeting agenda).  The number of genes combined in several of the talks was itself remarkable.  Medical applications are clearly coming down the pike.

Yet I found something lacking.  As in 2004, there was no mention this year of a critical set of tools required in any engineering field.  It may not be sexy, but test and measurement gear is what allows rapid comparison of prediction and experimental outcome.  Without sophisticated test gear, you have no Pentium, no 777, no Honda Element, no SpaceShipOne.  At the moment, while each experiment presented at SB 2.0 may be technically beautiful and impressive, they are primarily one-offs.  There is no common signal, and there is no common way to compare experiments in different organisms.  This will eventually be addressed through some sort of standardization, such as is being attempted with Biobricks.  Yet I have always found the common signal in the Biobricks standard to be confusing.  I forget what it was called originally, but now the input and output relationships of the parts are defined in Polymerases Per Second, or POPS, the number of polymerases running into, or out of, a genetic element in a second.

As I write this, I finally realize why I don't like POPS.  As Drew Endy describes it, POPS is a way to allow abstraction from the level of genes and specific proteins up to devices with a common reference.  I understand this story, and it makes sense to me given the constraints of the biological parts we have to work with.  But here's the thing: measuring POPS is presently exceptionally hard.  You can test each part in a framework that allows the measurement of POPS, probably using a fluorescent protein as an output signal, which is only vaguely quantitative.  It is also not a direct measure of POPS, as there is at least one layer of function between the number of RNA polymerases running down DNA and the number of proteins that get translated from RNA.   But it gets worse; how to you troubleshoot the entire circuit?  Where do you stick the multimeter probes on the fly to see why your circuit isn't behaving as expected?  You don't.  Instead, you resort to microarrays to check RNA expression levels or you use protein assays.  Until there is a magic "POPSometer", there won't be any way to examine a circuit in real time.  Fluorescent proteins will never adequately fill this role, 1) because of the time required to fold and produce a fluorescent signal and 2) because you have to build a new circuit every time you want to stick the test probe in a new spot.

Moreover, tools presently in use provide the illusion to the uninitiated that the physical infrastructure of synthetic biology is already well developed.  It is fairly straightforward to get single cell fluorescence or behavior data at this point, but you have to presume the organism is running the program you wrote.  Separately, it is easy to sequence large amounts of DNA, generally purified from many individuals.  But you can't yet sequence a given bug behaving in a given way to make sure it is following the DNA you put into it.  And readily available sequencing technologies average over variation present in a population that may be critical to understanding function.

This technological mismatch extends to discussions of security.  We heard descriptions of various programs to monitor DNA synthesis efforts, which would tie into a surveillance network using a microbial background signal for the environment.  The later would serve as a reference for efforts to detect novel, and perhaps threatening organisms, in real time.  But there isn't yet any technology that can provide that sort of environmental information, nor will one be available in short order from what I have seen.

In summary, we are still at the beginning of a very long road.  Before chemical engineering came synthetic chemistry, and before biological engineering will come synthetic biology.  I just wish the community had better perspective on how far we have to go.

Synthetic Biology 2.0, Part IV :: What's in a name?

The last session at Synthetic Biology 2.0 was full of hand-wringing about the very name of the thing.  "Synthetic" seems to conjure up too many bogeymen for the likes of of many attendees.  The arguments against the name were all centered around the fact that "synthetic" is un-PC these days.  Never mind that we live in a world consisting entirely of synthetic food, clothes, houses, computers, solar panels, windmills, and liquid fuels.  Synthetic is just bad, evidently.

This debate is essentially about politics.  It seems the new field is scaring people just by it's name.  So perhaps we should choose a new name in order to finesse the acceptance of the science and technology?  After all, why fight more battles than you need to?

Okay, fine.  Go ahead and try to rename it.  I'll just watch this time, thanks.  Besides, I think the present name is both appropriate and inevitable, but more on that in a moment.  We started with a different name, once upon a time, and that one didn't go over so very well either.  In 2000, while trying to describe the way biology was about to change (here is the PDF), or at least the way Drew Endy and I were conceiving of a new biological engineering, I floated the phrase "Intentional Biology."  The text on that web page was last modified in late 2000, but the story is basically the same today.   Through predictive design, biological systems should be both easier to understand and more useful.  These engineered systems would behave as intended, rather than displaying random and mystifying behaviors often encountered when genetically modified organisms are introduced into new environments or set loose in the wild; i.e., unintended behaviors.  Roger Brent, Drew, and I, even organized a meeting to figure out how to make this happen.  "After the Genome 6, Achieving an Intentional Biology", was held in Tucson, AZ, in December of 2000.  Alas, that name had unintended consequences, namely that the biologists attending the meeting thought we were asserting that all prior molecular biology had been unintentional.  If rotten vegetables had been available, I'd have been pelted during my talk.

Not the best start.  Can't win them all.  A good lesson, too.

Fast forward to mid 2001 or so, when Drew and I are at a cocktail party in San Fransisco thrown in celebration of the opening of the new local office for Nature.  We wind up in a conversation with Carlos Bustamante, who regales us with the origin of the field of Synthetic Chemistry, and how this gives us the name for Synthetic Biology.  Drew and I are convinced.  But, of course, it wasn't up to Drew and I to name a new field.  We were simply looking for a name to distinguish what we wanted to do from how things had been done previously.  The phrase "Synthetic Biology" certainly isn't new, and was emerging from other sources at the same time (Steven Benner, in particular, if memory serves).

Drew has flirted with other names in the last 5 years, among them "constructive biology" and "natural engineering".  Craig Venter insists on calling it Synthetic Genomics.  Frankly, these aren't any more compelling to me than Synthetic Biology, and they also seem to require even more explanation.  At this point, I don't really care what it is called.  The work is going to happen regardless, and there is no way to turn back.  The name is only a lightening rod for criticism because, as Oliver Morton and others have pointed out, the community keeps drawing attention to itself and all the bad things it might facilitate.  But where is the good news?  I have tried in this space to point out the connections between Synthetic Biology and vaccines, to the possibility that Synthetic Biology might be our best hope to beat a pandemic, but it appears most people want to focus on the negative aspects of rapid and distributed DNA synthesis.  The recent SB 2.0 meeting started with a focus on biological production of energy, another excellent beneficial application, but any subsequent optimism was lost by the third day.

Now onto why the name is inevitable.  What we are doing has been called Synthetic Biology for almost a century.  Here is some text from my book:

Ch 4: 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. [Auth. note: Clear if you've read the first three chapters of the book, anyway.]

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 theoretical 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.

Thus the present project is truly different than the biology that has come before.  Synthetic Biology is based on an explicit reliance upon mathematical models.  My own particular bent here is in developing technology that enables better measurement of biological systems so as to test and constrain models and also to provide required capabilities for biological engineering.  Without that, we are stuck with Charles Davenport's criticism of seventy years ago.

"Synthetic Biology" fits, both linguistically and historically.  Why are we stuck on this same damn topic two years after the first meeting?  We have better, and more important, things to worry about.  And lot's of work to do.  Synthetic Biology 3.0 will take place in Zurich, Switzerland, 24-27 June, 2007.

Live from Synthetic Biology 2.0, Part III

Wandering out into the lobby, I found Paul Rabinow and Oliver Morton chatting about the future of DNA synthesis companies.  Oliver is blogging the meeting at his site Mainly Martian.

Earlier I mentioned the new flavor of money at this meeting and the presence of competing DNA synthesis companies.  Oliver is hot onto the story that these companies are already struggling with the fact that large scale synthesis is becoming commoditized, and they may not all be around for long.  (By the way, we are wondering why John Mulligan, founder of Blue Heron and an early entrant into the commercial synthesis game, isn't at the meeting.  His is a conspicuous absence. (UPDATE:  John Mulligan was on a camping trip and is apparently on his way here now.  But we still missed him yesterday.))  It seems like there is already quite a lot of pressure for desktop DNA synthesizers.

David Baltimore is speaking now about engineering the immune system, which I should tune into.

Live from Synthetic Biology 2.0, Part II

This year's meeting has an interesting new flavor, namely that of money.  There are VC's here (yesterday at lunch gave us the interesting sight of Vinod Khosla and Craig Venter sitting off together in a corner, no doubt planning the future of Synthetic Biology); the list of sponsors is heavy with corporate names.  This is all a great change from SB 1.0, which had a very academic feel.

Yesterday's "Synthesis Panel" was in fact a series of tag team marketing pitches from synthesis company executives, presumably in exchange for their sponsorship of the meeting.  The summary of that session, perhaps unintended, was that all four companies essentially gave quotations to the audience for synthesis jobs: "no more than four weeks, perfect synthesis, buck a base."  We also heard that they are expecting the cost curves to keep up the current pace, and that this time next year synthesis of genes will be $.50 a base.  We heard some discussion of changes in technology, but everybody is still essentially using the same chemistry, just different plumbing.  The presentation from Codon Devices included references to a bunch of interesting methods, including something I predicted/hoped would happen, namely the combination of the synthesis strategy published by Tian, et al., with the MutS purification scheme from Peter Carr at MIT.

The commercial (as opposed to governmental or foundation) money here is an indication that biological technologies are achieving recognition as a significant potential influence on the economy.  I still don't understand how to finesse the IP issues -- I've been working on a blog post and book chapter about "The State of Open Source Biology", or perhaps just "Open Biology", which just aren't ready for release yet.

Carolyn Bertozzi (UC Berkeley) is speaking now, which reveals another interesting thread to this meeting.  Prof. Bertozzi is presenting work on modifying extracellular sugar groups to better understand cell signaling and hopefully get at cancer diagnostics and therapeutics.  People are really waking up to the possibilities of combining powerful biochemistry with synthetic methods for building new pathways with exceptional power and flexibility.

Jack Szostak (Harvard) just stepped up to the microphone to speak about a "Model of Synthetic Protocell."  His protocell is a simple replicating vesicle with it's own nucleic acid instruction set, but he doesn't want to use any preexisting biochemical machinery.  "All processes must be spontaneous".  That's ambitious.  He says he doesn't think the work has any particular practical application, but I suspect that is just a matter of time.

Indonesian H5N1 Outbreak Linked to Cats?

(Sitting in the Synthetic Biology 2.0 meeting, so this will be brief.)

Following up on my earlier reports and speculation (here, here and here) about the role of felines in spreading H5N1:

The Australian Broadcast Company is carrying an interview in which Andrew Jeremijenko, Project Leader of the Influenza Surveillance Studies for a US Naval Medical Research Group, suggests the outbreak in Indonesia may be directly related to infection in cats.

The article, entitled "Failed Indonesian bird flu response concerns experts", by Peter Cave, contains the following exchange:

PETER CAVE: Are you seeing mutations in the virus in Indonesia?

ANDREW JEREMIJENKO: Yes, that's a good question. We are seeing mutations in the human virus. We are not seeing that same mutation in the bird virus. And that's of great concern.

Basically, when you do an investigation of a bird flu case, you should try to find the virus from the human and match it up with the virus from the bird and find the cause.

Now, in Indonesia, the investigations have been sub-optimal, and they have not been able to match the human virus to the poultry virus, so we really do not know where that virus is coming from in most of these human cases.

PETER CAVE: Does it suggest it's going through an intermediary before it's infecting humans?

[Andrew Jeremijenko]: It's a possibility that we can't rule out. I think they really need to do a lot more investigations. So far the closest match we have to the human virus is from a cat virus. So the cat could be an intermediate. We really don't know what's happening yet.

Live from Synthetic Biology 2.0

I'm sitting in Synthetic Biology 2.0 at UC Berkeley.  Talks started off with energy applications, which is interesting.  Evidently there was also a big VC meeting in the last few days that focused on SB applications to producing energy.

Steve Chu (Nobel Laureate in Physics and Director of LBL) led off the talks with proposals that "excess" crop land in US could be used to "grow energy", by producing appropriate plants and methods via synthetic biology.  He mentioned that despite global population expansion by a factor of ~2.5 in the last 60 years, cultivated land has only increased by ~10-15% due to increases in productivity.  But Chu made no mention of the problem that we have trashed lots of crop land in the last 50 years, and it isn't obvious that we could use large amounts of land to grow energy given the state of the soil.  More importantly, he made no mention of where we would get all the water to grow those energy crops.

I am all for growing our energy sustainably, of course, but I don't think that terrestrial crops have a hope of being the right answer.  Best meme from Chu's talk was starting off with the most efficient engine design, figuring out the best fuel for that engine, then designing an organism to produce that fuel.  Cool.

Craig Venter is speaking now.  Lots on minimal genomes and looking at alternative pathways for directly producing energy.  Directly photosynthetic production of methane, etc.

More soon.

Avian Flu Catch-Up

Changing diapers is definitely a distraction from H5N1, but now the kid is zonked out and I have a chance to catch up a bit.

Chinese Domestic Flu Vaccine Production

Almost a year ago, I examined the implications of the lack of pandemic preparedness in Asia, particularly China.  The April 06 issue of Nature Biotechnology carried a news piece (Pubmed) that basically confirms part of what little I had been able to determine about Chinese domestic flu production capacity.  Not much new in the piece, but at least it allows me to point to a reputable news source instead of merely one of my blog entries.

Avian Flu in Felines

In early March, I was prompted by an AP story to wonder about the implications of a cat killed by H5N1 in Europe as soon as the virus showed up there (see, "Avian Flu as a Harbinger of Zoonotic Diseases").  Soon after, Declan Butler came out with a news story in Nature about this, and recently Nature carried a commentary by Albert Osterhaus and colleagues exploring the issue in more detail (Kuiken, et al., Nature 440, 741-742 (6 April 2006) | doi:10.1038/440741a).  This latter piece takes issue with the bland and unconcerned statements by the WHO and OIE that H5N1 in felines is uninteresting and, more importantly, has no influence on the spread or evolution of the virus.

Kuiken, et al., observe that fatal infections in cats are common in SE Asia and the Middle East, and:

Given the high number of infected cats in these areas, and considering their ability to excrete virus into their surroundings in sufficient quantities that transmission between cats takes place under both natural and experimental conditions (see below), cats could be more than a dead-end host for H5N1 virus.

...Apart from the role that cats may play in H5N1 virus transmission to other species, they also may be involved in helping the virus to adapt to efficient human-to-human transmission.

There isn't any evidence of this, to be sure, but it is a damn scary thought.  It is important to note that there isn't any evidence in part because we really aren't doing a very good job of looking.  Our environmental monitoring for zoonotic diseases is dramatically underfunded.  Bugs that kill humans often come from animals, and we are doing a piss poor job of understanding how and why pathogens make the jump.  (See my post "Nature is Full of Surprises, and We Are Totally Unprepared".)

More Damn Cladistics

The 27 April, 06 issue of Nature has a back-and-forth between Taubenberger, et al., and two groups disputing the assertion that the 1918 pandemic flu virus was avian in origin.  Gibbs and Gibbs assert that the virus was in fact a reassortant previously present in mammals.  If nothing else, they get this right; "In light of this alternative interpretation, we suggest that the current intense surveillance of influenza viruses should be broadened to include mammalian sources."  (The growing awareness of the effects of the virus on felines is evidence we should be doing more to monitor the virus in the wild.  Never mind that of the $1.9 billion recently pledged to prepare for a pandemic, exactly none was pointed towards better monitoring.)   Antonovics, et al., similarly, argue that Taubenberger and colleagues got the phylogenetic tree wrong and that the amino acid sequences of the RNA polymerase genes put the 1918 virus, "within...clades containing strains from other mammalian hosts."  They conclude:

By stating that the high pathogenicity of the 1918 virus is related to its emergence as a human-adapted avian influenza virus, the authors raise the possibility that an emerging avian strain could resemble the 1918 flu. This alarming implication, which is based on misinterpretation of the phylogenetic data, is completely unjustified and could seriously distort the public perception of disease risk, with grave economic and social consequences.

Fair enough, if Taubenberger, et al., have in fact come to the wrong conclusion.  Taubenberger and co-authors give what appears to be a comprehensive hearing to their detractors, and appear to answer the challenge well.  I think the best bit of their argument is as follows:

We have never maintained that the virus entered the human population in 1918: rather, as described earlier, our claim that it entered the human population "shortly" before the pandemic should be interpreted as 'at least several years before the pandemic', as stated in our discussion. The path that the precursors of the 1918 pandemic virus took before emerging in humans in 1918 remains unknown. Phylogenetic analysis on its own cannot definitively resolve the issue. As in previous analyses, we analysed the sequences of these genes for clues about their origins and found that the proteins encoded by the 1918 polymerase genes were avian-like in all cases.

I like this bit in part, of course, because I can understand it.

The argument about the origin of the virus seems half well-founded and well-measured molecular evolution, and half complete hand-waving.  (My interpretation may be influenced by the Cointreau on the rocks I am working on.  This is Uncle Sydney's drink, and I have been hoping it will rub off, though, admittedly, it doesn't seem to be working.  Yet.  Buy, hey, it's fun to try.)  I am frustrated by the cladistics story in part because there is no way to judge the merits of either side of the argument without delving into the details of not only the sequence variation and fundamental virology but also the statistical models used to generate phylogenetic trees.  This, I definitely don't have time for.  Nor, I suspect, does anyone else outside the field.  We simply have to watch the debate and see where it goes.

The real point, of course, is that we don't know where the virus came from.  It is still a mystery and will likely remain so.  Because of the evidence presented by Oxford, et al., I tend to side with Taubenberger.  It doesn't really matter, though.  Regardless of where the virus came from, the lesson today is that since we don't understand what happened before, we are completely unprepared for anything like it that may come in the future, as I have written about before.

More Vaccine Tiddlywinks

Unfortunately, as I briefly alluded in a recent post, the NIH doesn't seem to be doing much to prepare us for future outbreaks.  While the Institute has awarded just over a billion dollars to manufacturers to get cell-culture vaccine production up and running, this is simply a new way to make the old vaccine.  In interviews with those same manufacturers for the bio-era Avian Flu and Economic Impacts of Genome Design projects, they were quick to admit cell-culture production will get vaccines out the door in, optimally, four to five months instead of the six to eight month figure overused when discussing egg-based production.  This is a modest improvement, to be sure, and it is possible that cell-culture techniques can be used to produce more doses.  But this doesn't help make better vaccines.  If we used cell culture to produce the present reference vaccine, we would still be screwed because it seems very likely that the reference vaccine will be next to useless.  Where is the additional funding for alternative, or fundamentally new, vaccine technologies?

A news story in the 27 April edition of Nature offers some slight hope.  In, "Flu-vaccine makers toil to boost supply," Carina Dennis writes that, "More than a dozen groups are developing pandemic vaccines, testing a range of strategies to boost potency and production capacity."  Ms. Dennis follows with the suggestion that moving from split virus vaccines to whole virus vaccines.  That is, using intact virions instead of the standard vaccine in which the virus is disrupted with detergent.  An accompanying map shows worldwide efforts to develop new vaccines, though I note only one subunit project (Solvay) and one surface antigen project (Chiron).  There is no mention of DNA vaccines, either from PowderMed, delivered using gene guns, or from Vical, delivered via intramuscular injection, which I am waiting to hear back about from the manufacturer and from the doc in charge of a recent clinical trial.  Ms. Dennis notes that a study published by Neil Ferguson suggests that, "to curb the spread of disease, vaccinations would need to begin within one to two months of the pandemic starting."  Once again we are back to facing the need for a quick response while equipped with technology that is very slow.  And we have a crappy vaccine stock to start with.

And with that, I am tempted to start ranting again about the need to completely revamp our technological response to infectious disease, which you have all heard before.  So it is time to sleep and dream of better things.  Like changing diapers.

Comments on new FDA rules for expedited development of pandemic influenza vaccines

I'm a bit behind on the blog, having spent most of the last four weeks on the road, battling a norovirus (Yuck.  Don't get this bug  Hey Ralph, where the hell is my vaccine?), doing my taxes, and buckling down in the lab trying to get devices fabricated.  Cool progress on the later, which, alas, has to be disclosed to the University, written up for publication, and a patent application filed before I can discuss it here.  Harrumph.

A great deal has transpired on the influenza front, including news that the current H5N1 vaccine is as useless (NY Times) as predicted, which means that the need for alternative vaccine technologies is now even greater (more on this in a forthcoming post).  But I will start with the FDA's new draft guidelines on accelerated licensing of influenza vaccines, which I briefly addressed at the beginning of March.  The suggested guidelines were officially announced a few days later.  What follows is my take after a quick once over.

Here is how the document (PDF warning) starts off:

This document is intended to provide to you, sponsors of pandemic influenza vaccines, guidance on clinical development approaches to facilitate and expedite the licensure of influenza vaccines for the prevention of disease caused by pandemic influenza viruses.  The approaches apply to "split virus" and whole virus inactivated pandemic vaccines propagated in embryonated chicken eggs, and are also applicable to cell-culture derived, recombinant hemagglutinin-based protein, and adjuvanted pandemic influenza vaccines.  We, FDA, also address live attenuated influenza vaccines. This document does not address influenza vaccines that do not contain a hemagglutinin component.  Current U.S. licensed influenza vaccines are trivalent vaccines approved for the prevention of seasonal influenza illness.  Two classes of vaccines are licensed, "split virus" trivalent inactivated vaccines and a live attenuated trivalent vaccine. (pg. 1)

Beginning on page 2, by the way, is a short and very nice introduction to the biology and history of pandemic influenza viruses.

Here are some interesting quotes from the press release:

The FDA provides manufacturers with clear guidance on developing and submitting clinical data to show safety and effectiveness for new vaccines.  Consistent with the aims of FDA's Critical Path Initiative to get products to market more quickly and to advance the development and use of new technologies, these documents outline specific approaches that vaccine developers may follow.

...In issuing this advice, FDA aims to facilitate manufacturers in increasing the number of doses to ensure that enough influenza vaccine is available to vaccinate each person in the at-risk population. Having additional diversity in our vaccine supply helps enhance the capacity to produce more doses of influenza vaccine and contributes to the nation's pandemic preparedness.

...The release of these guidances is part of the comprehensive effort that FDA is undertaking to work with manufacturers to facilitate the development of vaccines.  Other examples include a recent CBER advisory committee meeting to discuss novel approaches to develop influenza vaccine such as using cell technology rather than eggs, frequent interactions with vaccine manufacturers to provide both scientific and regulatory guidance, as well as CBER's preparation of material for testing the potency of new vaccines, which are made available to manufacturers.

All in all, an exceptionally conservative document, given the magnitude of the problem we are facing.  The recommendations should do very nicely in all circumstances save the emergence of an actual pandemic.

The main problem I have with this announcement is that it appears to be geared towards easing the way for approval and use of particular technologies -- namely vaccine production by cell-culture, split virus vaccines, and specific recombinant protein vaccines -- rather than defining engineering and immunological goals that any given technology should meet to be approved.  For example, PowderMed's DNA vaccine does not appear to fit into the new guidelines, despite arguments the company is likely to make that injecting a plasmid coding for the hemagglutinin is equivalent to injecting the protein itself.  I hope I am wrong about this, because DNA vaccines look to be the only technology that can be used to respond on short time scales to rapidly spreading diseases.

No doubt I will return to this issue as the rules are discussed and developed.

Update on H5N1 Evolution

Chen, et al., ("Establishment of multiple sublineages of H5N1 influenza virus in Asia; Implications for pandemic control" PNAS) report that:

Genetically and antigenically distinct sublineages of H5N1 virus have become established in poultry in different geographical regions of Southeast Asia, indicating the long-term endemicity of the virus, and the isolation of H5N1 virus from apparently healthy migratory birds in southern China. Our data show that H5N1 influenza virus, has continued to spread from its established source in southern China to other regions through transport of poultry and bird migration. The identification of regionally distinct sublineages contributes to the understanding of the mechanism for the perpetuation and spread of H5N1, providing information that is directly relevant to control of the source of infection in poultry. It points to the necessity of surveillance that is geographically broader than previously supposed and that includes H5N1 viruses of greater genetic and antigenic diversity.

And also that:

Our ongoing influenza virus surveillance in southern China shows that H5N1 influenza viruses have been persistently circulating in market poultry populations and also revealed that those viruses were present in apparently healthy migratory birds just before their migration. Genetic analyses reveal that the endemicity of the H5N1 viruses in domestic poultry has resulted in the establishment of distinct regional virus sublineages. The findings of this study demonstrate that H5N1 viruses can be transmitted over long distances by migratory birds. However, viruses in domestic poultry have evolved into distinct regional clades, suggesting that transmission within poultry is the major mechanism for sustaining H5N1 virus endemicity in this region.

Because some migratory ducks sampled in the study have a stronger serological response to an H5N2 probe than to H5N1, it may be that prior infection with a low pathogenic H5 virus has provide some protection against H5N1.  That is, the ducks can carry H5N1 but display no symptoms.  While this is good for those particular ducks, unfortunately it means that rather than dying the ducks can easily transport H5N1 long distances to populations that are completely immune naive for H5 viruses.

The Chen paper also reports the worrisome result that an antiserum raised in ferrets against the current human H5N1 vaccine candidate was strongly reactive against isolates from Vietnam but only weakly reactive against isolates from Indonesia and large parts of China.  Conversely, a ferret antiserum raised against an Indonesian isolate reacted only weakly with isolates from Vietnam and China.  This means the antibodies prompted by the strains from Vietnam don't work against the Indonesian and Chinese strains, and vice versa.  So there is already considerable divergence of the sequence in the wild, and some sequences are not recognized by the present human candidate vaccine.

This becomes even more troublesome with the observation by Chen, et al., of a new genotype in the wild composed of pieces of previously seen ones.  Thus not only are the avian H5N1 strains diverging in the wild to the point that they do not cross-prime mammalian immune systems, but they are also actively swapping parts on time scales that we can now resolve.  It is excellent news that we can actually see what is going on, though not in real time, but this demonstrates that the viruses are clearly able to exchange useful innovations in short time scales, thereby producing new bugs.  The authors conclude from sequence data of the new genotype that, "all eight gene segments of viruses from the Qinghai Lake outbreak in central China can be traced to the H5N1 viruses isolated from migratory ducks at Poyang Lake in southeast China, ~1,700 km distant, indicating that migratory birds can disseminate the virus over long distances."

Chen, et al., conclude the paper with:

The antigenic diversity of viruses currently circulating in Southeast Asia and southern China challenges the wisdom of reliance on a single human vaccine candidate virus for pandemic preparedness; the choice of candidate viruses for development of human vaccines must reflect the antigenic diversity observed across this wider region. Furthermore, antigenic drift observed over time within those H5N1 sublineages highlights the necessity of continually updating the candidate virus chosen for future H5N1 vaccines. These concepts are critical for the control of this pandemic threat.

Which is right on the money, as far as I am concerned.  Except, of course, it would be nice to see more people banging the DNA vaccine drum.

Meanwhile, Stevens, et al., report in an upcoming Science article that, "The hemagglutinin (HA) structure from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA."  They also study specific mutations to various HA domains to gain insight into potential paths "for this H5N1 virus to gain a foothold in the human population."

The authors come to no specific conclusions about the likelihood of any given mutation, but using a recombinant system do identify a couple of changes that could lead to greater pathogenicity in humans.  A good step forward, though despite all the detailed biochemistry and molecular biology in this paper it leaves me once again feeling like we are still very poorly informed about basic flu virus biology and are simply guessing about the future course of the H5N1 in particular.