Taking Issue With Henry Niman

Henry Niman has made quite a lot of bother about the Avian Flu in the last year at his site Recombinomics.  It is still unclear whether he has a better handle on the future of the virus than does the WHO, the CDC, the FAO, and the UN, in part because he has yet to publish anything in a peer reviewed article.  But it is clear some folks are getting touchy about Niman's vocal assertions of doom -- here is a blog entry rebutting some of Niman's claims, and otherwise taking him to task for his behavior.  There's more, for those interested in this sort of thing, at drmartinwilliams.com.

UPDATE (24 October 2005):  Here is a previous post of mine that includes an attempt to figure out whether Niman has interesting or useful contributions to the Avian Flu problem, and a post attempting to sort out the difference, if any, between recombination and reassortment.  Also an early commentary on how little data we have about what evolutionary mechanisms result in pandemic flu strains.

UPDATE (1 November 2005):  Here is a post illustrating the gap between Niman's claims and conclusions based on sequencing data from the World Health Organization.

UN Bird Flu Chief Predicts 150 Million Deaths

When it comes to predictions of the number of deaths from H5N1, I tend to look skeptically at the numbers.  However, the UN has just appointed Dr David Nabarro as its Bird Flu chief, and the BBC world service is quoting him in their headlines as saying the toll will be as high as 150 million.  That is truly remarkable, and the interview with the BBC he says firmly "when" not "if".

PowderMed's H5N1 DNA Vaccine

The news service at Nature is reporting ("Bird flu vaccine not up to scratch" -- subscription required) that an egg-based whole virus Avian Flu vaccine, recently announced with fanfare as solving all our problems, is unlikely to be useful.  Fortunately, there is an alternative.  Alas, regulatory issues may prevent PowderMed from distributing it's DNA vaccine for the Avian Flu for some years yet.

The whole virus vaccine was announced with great fanfare just a few months ago.  But as I've written previously (here and here), egg-based vaccine production will never be sufficient for rapid responses to quickly evolving viruses like the flu.  Moreover, to produce a decent immune response the whole virus vaccine must be administered in 2 doses, each 6 times larger than an annual flu shot.  While this is in part because humans have never been exposed to an H5 virus (we are "immune naive"), it also appears that it just isn't a great vaccine.

While it is true that enthusiasm for DNA vaccines has gone through a bit of a boom and bust cycle, early results requiring high dose intramuscular injection are not representative of how the current technology works.  Genes coding for antigens for new viruses are slotted into a plasmid vector that has been proved safe in humans, the plasmids are loaded onto micron-sized gold particles, and the particles are injected into the skin using a high-pressure helium blast.  At Bio-ERA, we've been studying the vaccine and its utility, and it looks like the real deal.  An article in Red Herring quotes the CEO of PowderMed as saying;

What we really believe we’ve got is not just a vaccine; we actually have the ability to produce a capability for a country to cover anything really.  We have designed, with the help of contract manufacturers, a facility that would be able to produce 150 million doses in three months.

The key to the value of PowderMed technology is that the DNA vaccine is delivered directly in the nucleus of dendritic cells in the epidermis.  By getting dendritic cells to express coat proteins from pathogens and then present those proteins in complexes with MHC molecules, the vaccine directly stimulates a cellular immune response; T-cells are thereby primed to recognize and dispose of the virus and infected cells.

Vaccine production in chicken eggs or in cell culture requires at least 6 months to even begin cranking out doses, and requires significant infrastructure to do so.  PowderMed suggests that within three months of sequencing a new pathogen they can have vaccines ready to go.  But my estimates suggest it could be much faster than this.  Included in PowderMed's estimate is the time required to load the vaccine into their proprietary delivery system (a helium powered injector about the size of a flashlight).

My own estimate of the time required to fabricate the plasmids, followed by enzymatic amplification, is more like a week or two.  Injection of the vaccine does require particular technology (a "gene gun") but as it happens those have been used for ~10 years to genetically modify plants and animals.  There are gene guns scattered all across the developed and developing world.  If we had to, if the Avian Flu started to cause real problems in the human population, we could synthesize the vaccine in a widely distributed fashion (anywhere around the globe where people have access to large scale DNA synthesis) and deliver it using gene guns.  True, those instruments were intended for research use only, and were not designed for (or at least not marketed for) use on humans.  But if things start to go south, I'll be first in line.

The four stages of adopting new ideas.

Perhaps the best summary of life in academia that I have ever heard:

There are four stages of adopting new ideas

The first is “It’s impossible”
The second is “Maybe it’s possible, but it’s weak and uninteresting”
The third is, “It’s true and I told you so”
And the fourth is, “I thought of it first”

From H. Koprowski, "Vaccines and sera through plant biotechnology," Vaccine, 23 (2005), 1757-1763.

Synthetic Biology for HIV prevention: "A live microbial microbicide for HIV"

In the latest issue of Proceedings of the National Academy of Sciences, Rao et al demonstrate a fascinating, and probably immensely useful, application of genetic modification.  They altered human commensal strain of E. coli to excrete proteins that prevent HIV from infecting immune cells.

This isn't the first time bacteria have been genetically modified to carry antigens, antibodies, or, as in the present example, peptides that directly interfere with a pathogen's mode of infection.  But it is particularly interesting because the authors chose as a delivery strain a bug that is available as an over the counter probiotic supplement used to treat irritable bowel disease, cholitis, and Crohn's Disease.  The strain, "Nissle 1917", has thus already been demonstrated safe for use in humans, and is distributed in capsules intended for oral ingestion that can be easily manufactured and then stored at room temperature.

It's important to note that while this paper shows the bacterium prevents HIV infection in cell culture, and that the bacterium survives in the intestines of mice while secreting "inhibitory concentrations of the anti-HIV peptide onto mucosal surfaces of the gastrointestinal tract", it does not actually demonstrate prevention of infection in an animal model, let alone humans.  This experiment will no doubt require considerable review by institutional committees, and perhaps by the NIH itself (which is likely, since the work took place at the National Institute of Allergy and Infectious Disease).

Nonetheless, this is a quite sophisticated application of biological technology to address human needs.  The anti-HIV peptide is itself a synthetic product, being a fusion of hemolysin-A and a fragment of gp-41, the later chosen because it binds to the protein complex on HIV that enables it to dock with and then enter human cells.  Hemolysin-A served as the "shipping tag"; it is part of a protein excretion system already present in E. coli.  Thus this work demonstrates the modification of a bacterial strain -- one already known to be well tolerated in humans -- by exploiting an extant protein export system to excrete a synthetic protein cargo that may provide significant protection against HIV.

Like Jay Keasling's work to manufacture anti-malarial drugs in E. coli, Rao et al are on the path to producing organisms that can be easily and inexpensively grown in culture, followed by harvest and packaging of therapeutic compounds or of the bugs themselves.  This production work is similar to commercial efforts in India, China, and other developing countries, and helps pave the way to distributed biological manufacturing(PDF).  There is clearly lots of work left to do before humans are given genetically modified bacteria as preventative microbicides, but the world it is achangin'.

Cheap drug and stem cell trials in China

"Leaders and laggards in the stem cell enterprise," in the 30 June 05 issue of Nature Biotechnology, describes the global distribution of policy and science of stem cell research.  The story notes that;

The wild cards in all this are China and South Korea. Both have extremely talented scientists, but both are (perhaps unfairly) singled out for lax ethical standards and an uninformed public. With fewer shackles on the momentum of ES cell research, the two countries could potentially accelerate products into clinical trials much faster than the rest of the world.

But just because progress might be made in the clinic, it isn't clear that drugs (including cell-based therapies) produced in China will find an easy time in U.S. and European markets.

In "China beckons to clinical trial sponsors", Hepeng Jia writes that;

The clinical trials market is opening up in China...Low cost and ease of access to patients are the main incentives for clinical trial outsourcing...Ying Zhang, marketing director at Beijing-based clinical trial contract research organization (CRO) Excel Medical Technology, estimates that the cost of a clinical trial for a new drug in China is only half of the amount in the United States or Western Europe owing to lower labor and infrastructure costs.

There are, however, a few challenges, including, "poor data standardization, delays in gaining trial authorization from regulators and questionable ethical standards," and a, "poor level of compliance with Western standards."

Despite the harmonization of standards at some clinical trials facilities, it remains to be seen whether the US Food and Drug Administration or European Medicines Agency will accept data obtained from clinical trials carried out in China. Already foreign organizations like Quintiles, CCBR and Canadian company MDS of Toronto, have been seeking endorsement of agencies like the American College of Pathologists to gain recognition for their work there.

Large pharmaceutical corporations are moving into the Chinese market via acquisition.  Alla Katsnelson reports (Nature Biotechnology 23, 765 (2005)) that the Israeli generic manufacturer Teva Pharmaceuticals is buying Tianjin Hualida Biotechnology Pharmaceutical Co, in part because of "regulatory purgatory" in the United States.  "By tackling less strictly regulated markets first, Teva could gain more expertise in manufacturing biogenerics and profit from those markets directly."

Writes Katznelson;

The first problem is that products made in China are arguably unlikely to be approvable in Western countries [by the FDA and EMEA].  'Standards in the US are so much higher that there's just no comparison'.
    Getting non-Western facilities up to production standards is a long and expensive process...  For example, requirements for manufacturing specific such as water purity may not be important clinically, but the FDA is likely to insist on them.

Yet Teva is evidently pursuing a long-term strategy, because while the FDA has for several years been issuing assurances that, "creating a regulatory pathway for biogenerics is a top priority...its reactionary approach has not inspired confidence in the generics industry of late;"

RA used to mean regulatory affairs, but now it means risk aversion," says Alan Liss, senior director of biotech at Duramed Research in Pennsylvania. "While we're talking, China and India will be supplying the rest of the world with products," he adds.

But the story concludes that this same sort of transition occurred in India, that Indian drug development and manufacturing efforts were "eventually folded in the Western regulatory framework," and that "in ten years, companies will use China not only as a local market but as a springboard for global opportunities -- providing talent and infrastructure at a good price."

Gong Yidong, writing in the 29 July issue of Science, notes the many motivations for operating in China;

China's growing appetite for Western drugs--the current $15 billion market is expected to quadruple by 2010, and then double again by 2020--has certainly caught the attention of every drug company. So has its cheap but skilled scientific labor force. Not only do Ph.D.s receive annual salaries of $10,000 or less, but the most expensive aspect of drug development--clinical trials--costs an estimated 30% less in China than in the United States or Europe. And then there is its growing prowess in science. "I'd say that setting up our own research lab there is only a matter of time," Novartis CEO Dan Vasella remarked this spring. "It's not so much a need as it is a hunger to take advantage of the opportunities."

With many companies opening R&D labs in country, combined with the increasing level of education and the desire to take a larger role on the world state, we're likely to see remarkable ferment developing new treatments in China.

Biotech Oases -- your gas purchases at work.

Hot on the heels of news about serious biotech investment in Kazakhstan comes an update on similar efforts in the Middle East.  In the latest Nature Biotechnology, Cormac Sheridan describes (PDF only) initiatives in Dubai, Abu Dhabi, Qatar, and Saudi Arabia to nucleate biotech business and education.

Tax incentives, government sponsored facilities and infrastructure, and multi-billion dollar endowments for training and scientific grants are all part of efforts to bootstrap local technology development.  Strong, well-funded connections are being built with western educational institutions.  The story notes that countries in this region do not have a strong history (recently, anyway) of scientific research, and that part of the challenge will be to create a culture of innovation and competition to produce new results.  Oil money is paying for all of this, and I have to say that I am not unhappy some of the profits from filling my gas tank will go to this sort of investment.

In the long run, I wonder what will be the domestic social and political impacts of encouraging inquiry and increased contact with western scientists.  It's also worth asking, given historical Arab leadership in education and scholarship, about the local mores (Islamic or otherwise) regarding cloning, stem cells, genetic modification, and cell based therapies.  Does anyone have suggestions for reading along these lines?

Commentary on Flu Vaccine News and Epidemiological Models

I've been mulling whether to wade into the fray generated by recent reports of "effective" H5N1 vaccines and computer models that suggest a pandemic might be stoppable.  Fortunately, I see that the folks at EffectMeasure took care of most of what I wanted to say.  Basically, the media is distorting news of minimal (and possible irrelevent) progress on the vaccine to make it sound like we are all set and ready for whatever comes, while the models show that only if we are very lucky will anti-virals and quarantines slow a pandemic (the comments on both posts are worth reading, too).  I feel so much safer.

454's Microfabricated Pyrosequencer

Today, Nature published online an article entitled, "Genome sequencing in microfabricated high-density picolitre reactors," by Margulies et al.  The paper describes embedding beads coated with DNA in 1.6 million wells etched into the end of a fiber-optic slide, where the slide is produced by repeatedly folding and drawing a fiber-optic cable.  Each well serves as a reaction chamber for the sequencing-by-synthesis method known as Pyrosequencing.  The research utilized an instrument built by 454 Life Sciences.

My email is ringing off the hook today with questions about how this fits into my estimates of sequencing and synthesis productivity ("Carlson Curves").  Thanks for your interest, everyone.

A few comments.  The first thing to note about the article is that the authors state they sequenced "25 million bases, at 99% or better accuracy, in one four hour run."  So at 6.25 million bases per hour, they appear to be doing quite well compared to a Sanger-based 96-capillary instrument, which the authors assert reads out 67,000 bases per hour.

Digging into the text a bit, we find that the average length of the DNA the authors were able to read was about 100 bases, which they note is far shorter than the ~750 bases standard in Sanger sequencing.  The article also notes that prepping the DNA samples required 10 person-hours; 4 hours for fragmenting genomic DNA into bite-sized pieces and generation library from those pieces, and 6 hours to put that DNA on beads and then put the beads on the sequencing chip.

So, that's roughly 14 hours from start to getting sequence data, which puts the productivity number at about 10 million bases per person per day.  This is better than running a couple of capillary-based instruments, it's true, but there is still an enormous amount of skilled labor in that 10 hours of sample preparation.  If you have look at the supplementary information, documents s1 and s3 in particular, the processing is by no means trivial.  Actually, the enzymatic rigmarole is quite impressive.  But I wouldn't want to do it myself.  Looking ahead, I don't see any reason it can't be automated.  Given time, patience, and some effort at the microfluidics, the whole process should require only minimal human attention.  That will definitely make an impact on productivity.  No doubt 454 is planning for this eventuality.  The upshot is that this paper puts a point, more-or-less, right on my previously published curves.  It is consistent with progress made with previous technologies, but is actually a bit slower than the estimate Mostafa Ronaghi gave me in 2003.  That's life.

Here's a bit more info.  The New York Times is reporting that;

Jonathan Rothberg, board chairman of 454 Life Sciences, said the company was already able to decode DNA 400 units at a time in test machines. It was working toward sequencing a human genome for $100,000, and if costs could be further reduced to $20,000 the sequencing of individual genomes would be medically worthwhile, Dr. Rothberg said.

We'll see.  We are still a long way from the Thousand Dollar Genome, and this paper appears to be keeping the pace.  All in all, it looks promising, though I wince at the current $500,000 instrument cost.  I don't have enough information at hand to make my own estimates of per base sequencing cost, and I haven't had a chance to contact anyone at the company to suss out the productivity issues better.  I'll update this if and when such conversations take place.

UPDATE (5 Aug 05):  The $500,000 per instrument cost comes from the NYT article:

The Joint Genome Institute, a federal genome sequencing center in Walnut Creek, Calif., has ordered one of 454's $500,000 sequencing machines but has not yet installed it. Paul Richardson, the institute's head of technology development, said the new approach "looks very, very promising" and could reduce sequencing costs fourfold.

The machine's limitation is that at present it can only read DNA fragments 100 units or so in length, compared with the 800-unit read length now attained by the Sanger-based machines. The shorter read length makes it harder to reassemble all the fragments into a complete genome, Dr. Richardson said, so although microbial genomes can be assembled with the new method, mammalian genomes may be beyond its reach at present.

Dr. Fraser, director of the Institute for Genomic Research in Rockville, Md., also said that the new machine's short read lengths "limit its overall utility at this point."

Global Distribution of Commercial DNA Foundries

(UPDATE, 22 November 05: Wired Magazine has now published a version of this map.)

Given recent discussions in the press and at the NSABB meeting concerning licensing DNA synthesis instruments and related professional skills, it seems like a good idea to make an estimate of how big the problem is by assessing the distribution of the technology.  Prompted by Jerry Epstein at the Center for Strategic and International Studies, I headed out on the web to make a list of Commercial DNA Foundries.  Here is a map we came up with to represent access to commercially synthesized oligos.

(UPDATE, 19 July 05: I've replaced the .gif with a higher resolution .jpg.)  (UPDATE: Note that these are Foundries -- that is, the building where DNA actually gets synthesized -- and that the associated distribution/marketing networks are actually considerably more widespread.)

This is just a first pass, though given how many companies there are I don't know if we will spend a lot of time trying to be encyclopedic.  A few notes:  there are no academic foundries on here, save the Zelinsky Institute in Moscow (which I included because it is quite interesting that a government facility in Russia is operating commercially -- fascinating implications for proliferation).  The number of academic foundries suggests that both instrumentation and skills are quite widely distributed.  The companies are numerous enough.  I gave up trying to fit more companies into the maps of US and Western Europe -- if I left out your company, my apologies.  Perhaps if we figure out a more clever way to keep track of, and represent, all the data, we can include all comers.  I suspect there are more companies in Russian and China, but the language barrier defeats my first pass with Google.

So now, a couple of thoughts.  The net capacity of all these foundries looks to be pretty impressive (though I have yet to add it all up).  Who is ordering all this DNA?  The estimates I've heard for the size of the synthesis market are in the low tens of millions of dollars annually.  Either many companies are ekeing out existence on wee small pieces of the total, or the market is much bigger than people think.  How is it split between short oligos, perhaps primarily used as PCR primers, and larger constructs used to build genes for recombinant proteins?  Does it make a difference, even now?  If so, given the increasing capability demonstrated in assembling short pieces of DNA, is it worth trying to distinguish between short and long oligos?  That is, will regulation of either short or long pieces of DNA be feasible and will it increase security?

Finally, I haven't yet charted the cost per base of synthesis as a function of geography, but I'm sure the results will be provocative.  I was surprised to see that the biotechnology industry in India is supporting at least three commercial synthesis foundries, and I'll bet those companies are charging less than I recently paid for gene synthesis domestically.  How soon are North American and European DNA foundries going to have to compete against Indian labor and FedEx?

More to come as I ponder this.  Comments and suggestions?