Tamiflu-resistant Influenza Strains

(Update, 30 April 2009: I see from the server logs that this post is getting a lot of traffic today.  Please note that the contents of the post discuss the annual influenza strains in the US, not the "H1N1 Influenza A" strain, which at this time is susceptible to Tamiflu.)

The IHT is carrying a great article by Donald Mcneil on the sudden emergence of antiviral resistance in this year's circulating influenza viruses.  The title says it all: "Flu in U.S. found resistant to main antiviral drug".

Virtually all the flu in the United States this season is resistant to the leading antiviral drug Tamiflu...  The problem is not yet a public health crisis because this has been a below-average flu season so far and the chief strain circulating is still susceptible to other drugs.

There are two important points in this story.  First, the resistance seems to derive from a spontaneous mutation rather than having emerged from overuse of the drug:

"It's quite shocking," said Dr. Kent Sepkowitz, director of infection control at Memorial Sloan-Kettering Cancer Center in New York. "We've never lost an antimicrobial this fast. It blew me away."

The mutation appears to have arisen in Norway, a country that the article suggests does not even use Tamiflu. Second, while the CDC is recommending that hospitals test all flu cases to find out whether patients are carrying a the resistant subtype, this capability is still not widespread:

"We're a fancy hospital, and we can't even do the ... test in a timely fashion," Sepkowitz said. "I have no idea what a doctor in an unfancy office without that lab backup can do."

I haven't written very much about the flu for a couple of years, but it is clear that the threat is still quite present.

The article ends with this bit of speculation:

And while seasonal flu is relatively mild, the Tamiflu resistance could transfer onto the H5N1 bird flu circulating in Asia and Egypt, which has killed millions of birds and about 250 people since 2003. Although H5N1 has not turned into a pandemic strain, as many experts recently feared it would, it still could -- and Tamiflu resistance in that case would be a disaster.

I'm not so sure that the resistance gene "could easily transfer onto the H5N1 bird flu".  It sounds like Mr. Mcneil may be giving more weight here to Henry Niman (who is quoted extensively in the article on other specific topics) than the rest of the community might.  This is not to say that such a transfer is unlikely -- this is the sort of thing that I fear we know so little about that we could make poor assumptions leading to even worse policy.  The mechanisms for recombination and reassortment of genes in the flu are still disputed in the literature.  But it's damn scary, either way, even if the probability of such a transfer is small.

In the end, if nothing else, what this demonstrates is that our technological base for both detecting and responding to infectious disease is still poorly developed.

iGEM 2008: Surprise -- The Future is Here Already.

I'm back from a weekend at MIT serving as a judge for the International Genetically Engineered Machines Competition.  Here are a few thoughts on the competition.

The "international" flavor continues to strengthen.  Of the six finalists, three were from the U.S., two from Europe, and one from Asia.  There were 85 teams registered, almost all of whom showed up.  I was hoping for more biofuels/energy projects, but perhaps that fad is already past.

The top three teams were (here are the full results): 1) Slovenia 2) Freiburg 3) Caltech.

First, a couple of slightly blurry iPhotos (when the hell is Apple going to upgrade that camera?):

IMG_0138 Tom Knight receives the BioBrick from the 2007 winner, Peking University.

IMG_0140 A collective dance party while the competitors wait for the judges.

IMG_0141 Tom Knight awards the BioBrick to the 2008 winners, Slovenia.

Several of the 2008 projects implement ideas that have appeared in science fiction stories and in my own speculations about the future of biological technologies:

UCSF characterized a fusion protein that enables epigenetic control of gene expression through chromatin silencing.  This, in effect, gives the user (which could be the cell itself) a new control knob for building memory circuits in eukaryotes.  I seem to recall that this is the basic innovation in Greg Bear's Blood Music that brings about the end of the world through Green Goo.  Go UCSF!

Caltech and NYMU-Taipei (check out the killer Wiki) both modified commensal E. coli strains to serve as therapeutics.  Caltech built a bunch of new functionality into the probiotic strain Nissle 1917, including microbicidal circuits, Vitamin B supplements, and lactase production (big kudos to Christina Smolke, here).  Taipei built a "Bactokidney" for people with kidney failure: cells that attach to the lining of the small intestine and absorb nasty substances that would otherwise need to be removed via dialysis.  These are both very cool ideas.

Seeing these projects brought back shades of a scenario published in Bio-era's "Genome Synthesis and Design Futures: Implications for the U.S. Economy".  (I wrote the original story, which was less complicated but slightly more nefarious than the Bio-era version, in 2005 as a short, provocative piece of a larger report for a TLA -- a three letter agency.)  Almost all the technology described below has been published in bits and pieces -- fortunately, it has not yet been put together in one microbe.

In 2008, the North Korean government launches a secret program to develop biological tools that can be used to pacify target populations for crowd control or military purposes. North Korea's research draws on Soviet work on modifying pathogens to express mood-altering peptides, and the demonstration by U.S. scientists at the National Institutes of Health that common commensal strains of E. coli could be modified to secrete specialized peptides in human intestines.  Modifying the same strain used by the NIH, available in an over-the-counter probiotic pill, the North Koreans secretly produce an organism that produces peptide hormones easily absorbed through the intestinal wall.

With further modifications to allow the peptides to enter the brain, the new strain produces a calming, almost sedative, effect on colonized individuals. Combined with a genetic circuit that confers both antibiotic resistance and upregulation of the peptides upon exposure to a chemical that can be dispersed like teargas, these modifications enable the government to pacify crowds in times of crisis. The E. coli can be distributed via food and water to target populations.

To maintain the presence of the genetic circuit within the population, the new strain is equipped with an antibiotic resistance mechanism from V. cholera that causes plasmids containing the entire genetic circuit, including the regulatory genes and the mood modification genes, to be horizontally transferred to other bacteria upon treatment with common antibiotics.

In 2009, Pyongyang uses military forces to suppress a widening political uprising against the regime. Reports of a "pacifying gas" quickly emerge, raising allegations about the use of chemical weapons. U.S. intelligence agencies claim that North Korea has used a novel combination of biological and chemical weapons against rioters, leading the U.S. to declare that Pyongyang has violated the international treaty on bioweapons. Pacifist biohackers undertake to recreate the microbe , or to invent new versions to use as "peace weapons" against armies.

When a U.S.-led coalition attempts to impose an economic embargo against North Korea, the Chinese government uses its military to secure supply lines to North Korea. A military standoff between U.S. and Chinese forces ensues.

Here is the original inspiration: "Toward a live microbial microbicide for HIV: Commensal bacteria secreting an HIV fusion inhibitor peptide". (I'd completely forgotten that I blogged the original paper.)

Slovenia won (again) with "Immunobricks" by engineering new vaccines. The technology they used forms the basis of arguments about rapid, distributed vaccine production we made in Genome Synthesis and Design Futures (Section 4.3, in particular), which I've also written about extensively here on this blog, and which will show up in my book.  Yet all of a sudden it's real, all the more so because it was an iGEM project.

From Slovenia's Wiki abstract:

Using synthetic biology approaches we managed to assemble functional "immunobricks" into a designer vaccine with a goal to activate both innate and acquired immune response to H. pylori. We successfully developed two forms of such designer vaccines. One was based on modifying H. pylori component (flagellin) such that it can now be recognized by the immune system. The other relied upon linking H. pylori components to certain molecules of the innate immune response (so called Toll-like receptors) to activate and guide H. pylori proteins to relevant compartments within the immune cell causing optimal innate and acquired immune response. Both types of vaccines have been thoroughly characterized in vitro (in test tubes or cells) as well as in vivo (laboratory mice) exhibiting substantial antibody response. Our strategy of both vaccines' design is not limited to H. pylori and can be applied to other pathogens. Additionally, our vaccines can be delivered using simple and inexpensive vaccination routes, which could be suitable also in third world countries.

If you've read this far into the post, you should definitely spend some time on Slovenia's Wiki.

Here's the short, pithy version: There is presently no vaccine for H. pylori.  Between June and October this year, seven undergraduates built and tested three kinds of brand new vaccines against H. pylori.  (They also put a whole mess of Biobrick parts into the Registry, which means those parts are all in the public domain.)

Yes, yes -- it's true, getting something to work in a mouse and in mammalian cell culture is a long way from getting it to work in humans, or even in ferrets.  But the skill level and speed of this work should make everyone sit up and take notice.

So it is worth pondering the broader implications of these projects.

The Slovenian team clearly has access to very high quality labs and protocols.  Mammalian cell culture can be very fiddly unless you know what you are doing and have the right equipment (I speak from painful experience, lo those many years ago in grad school).  The Caltech and Taipei teams also clearly have a great deal of support and mentoring.  Yet while bashing DNA and growing E. coli are not particularly hard, the design and testing of the coli projects is very impressive.

Despite all the support and money evident in the projects, there is absolutely no reason this work could not be done in a garage.  And all of the parts for these projects are now available from the Registry.

Over the past couple of years, in various venues, I have tried to point out both the utility and inevitability of proliferating biological technologies.  iGEM 2008 drives home the point yet again.  In particular, the ability to rapidly create vaccines and biological therapeutics points the way to increased participation by "amateurs", whether the professionals (and policy makers...and security types) are ready or not.  I'm also thinking back to "peer reviews" in which I was excoriated for suggesting this kind of work was within the reach of people with minimal formal training.  Because, really, you need a PhD, and an NIH grant, and tenure, to even think of taking on anything like a synthetic vaccine.  Oh, wait...

Although I've predicted in writing that this sort of thing would happen, I frankly expected practical implementation of both the rapid, synthetic vaccines and the modified commensal bacteria to take a few more years. Yet undergraduates are already building these things as summer projects.

It didn't really hit me until I started writing this post earlier this afternoon, but as I ponder the results from this year's iGEM only one thought comes to mind: "Holy crap -- hold on to your knickers."

The world is changing very, very quickly.

Update on Influenza Subtype Activity in 2007-2008

While addressing some comments from Ralph Baric on one chapter my book, I had reason to go find statistics on influenza subtype activity last year.  Those interested in keeping up on recent flu activity should peruse this July, 2008, report from the CDC: Influenza Activity --- United States and Worldwide, 2007--08 Season.

Here is the breakdown on subtype activity:

During September 30, 2007--May 17, 2008, World Health Organization and National Respiratory and Enteric Virus Surveillance System collaborating laboratories in the United States tested 225,329 specimens for influenza viruses; 39,827 (18%) were positive. Of the positive specimens, 28,263 (71%) were influenza A viruses, and 11,564 (29%) were influenza B viruses. Among the influenza A viruses, 8,290 (29%) were subtyped; 2,175 (26%) were influenza A (H1N1), and 6,115 (74%) were influenza A (H3N2) viruses. The proportion of specimens testing positive for influenza first exceeded 10% during the week ending January 12, 2008 (week 2), peaked at 32% during the week ending February 9, 2008 (week 6), and declined to <10% during the week ending April 19, 2008 (week 16). The proportion positive was above 10% for 14 consecutive weeks. The peak percentage of specimens testing positive for influenza during the previous three seasons ranged from 22% to 34% and the peak occurred during mid-February to early March. During the previous three influenza seasons, the number of consecutive weeks during which more than 10% of specimens tested positive for influenza ranged from 13 to 17 weeks.

Of note, 26% of samples positive for influenza were the H1N1 subtype -- the same as the 1918 flu -- which means we all have probably been exposed to it and have some immunity.  That does not mean the particular combination of genes in the 1918 flu would be harmless if it showed up again, but rather than our immune systems should be able to better recognize that bug and thus might defend against it better than the first time around.

Synthetic Biology 4.0 – Not so live blog, part 1

What a difference a few years makes.  SB 1.0 was mostly a bunch of professors and grad students in a relatively small, stuffy lecture hall at MIT.  SB 2.0 in Berkeley expanded a bit to include a few lawyers, sociologists, and venture capitalists.  (I skipped 3.0 in Zurich.)

At just over 600 attendees, SB 4.0 is more than twice as big as even 3.0, with just under half the roster from Asia.  The venue, at the Hong Kong University of Science and Technology, is absurdly nice, with a view over the ocean that beats even UCSB and UCSD.  Kudos also to the organizers here.  They worked very hard to make sure the meeting came off well, and it is clear they are interested in synthetic biology, and biotech in general, as a long term proposition.  The Finance Minister of Hong Kong, John Tsang, spoke one evening, and he was very clear that HK is planning to put quite a lot of money and effort into biology.

Which brings me to a general observation that Hong Kong really cares about the future, and is investing to bring it along that much sooner.  I arrived a day early in order to acclimate a bit and wander around the city, as my previous visit was somewhat hectic.  Even amid the financial crisis, the city feels more optimistic and energetic than most American cities I visit.

I will have to write up the rest of the meeting when I get back to the States later this week.  But here are a few thoughts:

As of the last few days, I have now seen all the pieces necessary to build a desktop gene printer.  I don’t have prediction when such a thing will arrive on the market, but there is no doubt in my mind that it is technically feasible.  With appropriate resources, I think it would take about 8 weeks to build a prototype.  It is that close.

Ralph Baric continues to do work on SARS that completely scares the shit out of me.  And I am really glad it is getting done, and also that he is the one doing it.  His work clearly demonstrates how real the threat from natural pathogens is, and how poorly prepared we are to deal with it.

Jian Xu, who is better known for his efforts to understand the human gut microbiome, spoke on the soup-to-nuts plant engineering and biofuels effort at the Qingdao Institute of Bioenergy and Bioprocess Technology, run by the Chinese Academy of Sciences (QIBEBT).   The Chinese are serious about putting GM plants into the field and deriving massive amounts of energy from biomass.

Daphne Prauss from Chromatin gave a great talk about artificial chromosomes in plants and how they speed up genetic modification.  I’ll have to understand this a bit better before I write about it.

Zach Serber from Amyris spoke about on their biofuels efforts, and Amyris is on schedule to get aviation fuel, diesel, and biogasoline into the market within the next couple of years.  All three fuels have equivalent or better characteristic as petro-fuels when it comes to vapor pressure, cloud point, cetane number, octane, energy density, etc.

More soon.

"Laying the foundations for a bio-economy"

My new commentary, "Laying the foundations for a bio-economy", will be appearing in a upcoming issue of Systems and Synthetic Biology.  The piece is freely available online as both text and PDF.  Thanks to Springer for supporting the Open Access option.  Here are the abstract, the first two paragraphs, and the last two paragraphs:

Abstract  Biologicaltechnologies are becoming an important part of the economy. Biotechnology already contributes at least 1% of US GDP, with revenues growing as much as 20% annually. The introduction of composable biological parts will enable an engineering discipline similar to the ones that resulted in modern aviation and information technology. As the sophistication of biological engineering increases, it will provide new goods and services at lower costs and higher efficiencies. Broad access to foundational engineering technologies is seen by some as a threat to physical and economic security. However, regulation of access will serve to suppress the innovation required to produce new vaccines and other countermeasures as well as limiting general economic growth.          

Welcome to the Paleobiotic Age. Just as today we look back somewhat wistfully on our quaint Paleolithic--literally "old stone"--ancestors, so will our descendants see the present age as that of "old biology", inhabited by Paleobiotic Man. The technologies we use to manipulate biological systems are experiencing dramatic improvement, and as a result are driving change throughout human economies.       

In order to understand the impact of our growing economic dependence on biological technologies it is worth taking a moment to consider the meaning of economy. "Economy" is variously thought of as, "the management of the resources of a country, especially with a view to its productivity" and "the disposition or regulation of the parts or functions of any organic whole; an organized system or method"  Amid a constantly increasing demand for resources, we look to technology to improve the productivity of labor, to improve the efficiency of industrial process and energy production, and to improve the yield of agriculture. Very tritely, we look to technological innovation within our economy to provide more stuff at lower cost. Biological technologies are increasingly playing that role.

...

In this, the Paleobiotic Age, our society is only just beginning to struggle with all the social and technical questions that arise from a fundamental transformation of the economy. History holds many lessons for those of us involved in creating new tools and new organisms and in trying to safely integrate these new technologies into an already complex socio-economic system. Alas, history also fails to provide examples of any technological system as powerful as rational engineering of biology. We have precious little guidance concerning how our socio-economic system might be changed in the Neobiotic Age to come. We can only attempt to minimize our mistakes and rapidly correct those we and others do make.

The coming bio-economy will be based on fundamentally less expensive and more distributed technologies than those that shaped the course of the 20th Century. Our choices about how to structure the system around biological technologies will determine the pace and effectiveness of innovation. As with the rest of the natural and human built world, the development of this system is decidedly in human hands. To paraphrase Stewart Brand: We are as engineers, and we'd better get good at it in a hurry.          

On Indonesia and Distribution of H5N1 Strains

News in the last couple of days that Indonesia has decided not to forward homegrown strains of H5N1 to the WHO and instead is dealing directly with Baxter Healthcare for production of vaccines.  The worst bit of this, of course, is that there does not appear to be much cross reactivity elicited by the Vietnamese and Indonesian isolates, where the international reference vaccine is derived from a strain isolated in Vietnam.  Moreover, while Baxter is supposedly making progress in producing influenza vaccines in cell culture (Baxter's Press Releases, CIDRAP's version), this technology is not yet approved for human use; only research contracts, rather than production contracts, have been let by the U.S. Government for cell culture production.  Finally, despite much noise that cell culture is faster/better/cheaper than eggs for producing vaccines, it appears cell culture only beats eggs by a month or two.  (Baxter does have a very comendably decent Influenza information web page, which is here.) 

Here are a few paragraphs from an AP story, "Experts say Indonesian deal on H5N1 virus jeopardizes race for pandemic vaccine", via the IHT:

Indonesia Wednesday signed a memorandum of understanding with U.S. drug manufacturer Baxter Healthcare Corp. to develop a human bird flu vaccine.

Under the agreement, Indonesia will provide H5N1 virus samples in exchange for Baxter's expertise in vaccine production. Other organizations would have access to Indonesian samples provided they agree not to use the viruses for "commercial" purposes, said Siti Fadilah Supari, Indonesia's health minister.

But that is a major departure from the World Health Organization's existing virus-sharing system, where bird flu viruses are freely shared with the global community for public health purposes, including vaccine and antiviral development. Indonesia has not shared any viruses since the beginning of 2007.

Indonesia defended its decision, arguing the system works against poor countries. "The specimens we send to WHO...are then used by vaccine makers who then sell to us (at a profit)," Supari told reporters Wednesday. "This is unfair, we have the virus, we are getting sick, and then they take the virus from WHO — 'with WHO's permission' they say — and make it themselves," said Supari.

There seems to be a bit of confusion among reporters about whether Indonesia now has an official policy of withholding samples from the WHO, but Baxter is making it clear they don't have anything to do with the decision.  From The New York Times' coverage: "A Baxter spokeswoman said the company had not asked Indonesia to stop cooperating with the W.H.O. She added that the agreement under negotiation would not give it exclusive access to Indonesian strains."

In any event, Indonesians feel bent out of shape that they have previously provided strains to the international community, only to be charged for the vaccine when it becomes available.  News reports portray this as something of an IP spat, akin to controversy over biomining.  From the Reuters coverage:

"The specimens we sent to the WHO have been forwarded to their collaborating center. There it has been used for various reasons such as vaccine development ... or research," Supari said.

"Later they sold the discovery to us. This is not fair. We are the ones who got sick. They took the sample through WHO and with WHO consent and they tried to produce it for their own use," she said at a news conference after the signing of the pact with Baxter.

Supari said Australia was producing a human bird flu vaccine using the Indonesian virus strain, but did not give details.

"I was shocked because I never gave permits to Australia to produce a vaccine using our strain," she said.

"We have been working with Baxter since the beginning and are processing intellectual property rights with them. Baxter protects our intellectual property rights," she said.

...Under the memorandum of understanding, Indonesia would have the right to produce and market the bird flu vaccine domestically. It is negotiating to export it to a number of countries.

Production would be carried out by makers appointed by the Health Ministry.

So, in conclusion, the deal appears to put Indonesian isolates of H5N1 out of the reach of governments and firms with other vaccine technologies, at least for the time being.  Finally, in an interesting twist on the distribution of biological technologies, the deal also appears to put Indonesia in a position to become a leader in cell culture production of vaccines, potentially jumping to the head of the pack in the international vaccine market.

H5N1 is back in the U.K.

The headlines are today loudly announcing the return of H5N1 to the United Kingdom (CNN, New York Times) at a Turkey farm near Lowestoft.  Though nobody can say for sure, the virus probably arrived via migrating birds.  It appears that the likelihood of transmission by migrating bird or smuggled poultry has a geopolitical dependence.

Last month, Kilpatrick, et al., published a paper in PNAS ("Predicting the global spread of H5N1 influenza") that looked at a variety of factors to classify historical outbreaks and predict new ones.  The abstract does a decent job of summarizing the paper, so here it is:

The spread of highly pathogenic H5N1 avian influenza into Asia, Europe, and Africa has resulted in enormous impacts on the poultry industry and presents an important threat to human health. The pathways by which the virus has and will spread between countries have been debated extensively, but have yet to be analyzed comprehensively and quantitatively. We integrated data on phylogenetic relationships of virus isolates, migratory bird movements, and trade in poultry and wild birds to determine the pathway for 52 individual introduction events into countries and predict future spread. We show that 9 of 21 of H5N1 introductions to countries in Asia were most likely through poultry, and 3 of 21 were most likely through migrating birds. In contrast, spread to most (20/23) countries in Europe was most likely through migratory birds. Spread in Africa was likely partly by poultry (2/8 introductions) and partly by migrating birds (3/8). Our analyses predict that H5N1 is more likely to be introduced into the Western Hemisphere through infected poultry and into the mainland United States by subsequent movement of migrating birds from neighboring countries, rather than from eastern Siberia. These results highlight the potential synergism between trade and wild animal movement in the emergence and pandemic spread of pathogens and demonstrate the value of predictive models for disease control.

Of course, the only way to know if the model really works is, alas, to wait for more outbreaks.  Anyway, it seems the U.S. is safe from poultry smuggling, which we have a chance of intercepting, but susceptible to migrating birds, a pathway that almost certainly resists any defensive measures.

 

H5N1 Influenza and Countermeasures Update

There is an excellent news piece in last week's Science, where here the definition of excellent is both "informative" and "highly unsettling".  Dennis Normile and Martin Enserink write:

An upsurge in H5N1 bird flu outbreaks in poultry across Asia is driving home the message that even countries that have eliminated the virus once shouldn't become complacent. The continuing high death toll in humans, including two recently detected cases of infection with a Tamiflu-resistant strain in Egypt, is also a grim reminder of how devastating the virus might be if it acquires the ability to spread easily among humans.

...Over the past 3 weeks, Thailand and Vietnam reported their first H5N1 outbreaks among poultry in 6 months. Japan, which seemed to have dodged the bullet since its cluster of outbreaks in 2004, confirmed that the virus hit one farm on 11 January and probably a second farm on the 23rd. South Korea, which last November suffered its first outbreak since containing the virus in 2004, reported that the virus had turned up on a fifth poultry farm. Several wild birds found dead in Hong Kong tested positive for H5N1. And Indonesia on 20 January reported its fifth human death from the virus in just 10 days, bringing its death toll to 62, by far the most of any country.

The increase in outbreaks in the Northern Hemisphere follows what has become an established pattern. The reason for the seasonality is still not well understood, says Les Sims, a veterinarian based in Manunda, Australia, who advises the U.N.'s Food and Agriculture Organization (FAO). It is likely to be some complex interaction among several factors, including cooler temperatures enabling the virus to survive longer in the environment, greater poultry trade in preparation for winter festivals, and movements of wild birds.

The recurrence of the virus in South Korea and Japan is particularly notable. In both the winter of 2003-'04 and this year, outbreaks in South Korea were followed 4 to 6 weeks later by outbreaks in Japan. "The outbreaks in Japan and South Korea suggest to me free-flying birds as the most likely origin," says Sims. Both countries are trying to determine how the virus was reintroduced.

So it seems unlikely we will be rid of the virus through culling programs, the primary mechanism thus far employed for biosecurity.  That the virus seems to be spread by wild birds in these cases is interesting, but this isn't the only pathway for reintroduction into poultry or people.

Last week's issue of New Scientist revisits the notion that "Deadly H5N1 may be brewing in cats".  (Most of the relevant text is available here at ProMed.)  Felines may be serving as a mammalian host that might enable the virus to adapt to mammalian biology and thereby become more dangerous to humans.  This is something I started wondering about after cats started dying in Europe so soon after the virus arrived there last year.  The New Scientist provides corroborating evidence that cats are important in the dynamics of the virus in Indonesia.  The story reports some surprise on the part of scientists doing the field work with regard to the prevalence of the virus in cats in Indonesia:

In the first survey of its kind, an Indonesian scientist has found that in areas where there have been outbreaks of H5N1 in poultry and humans, 1 in 5 cats have been infected with the virus, and survived. This suggests that as outbreaks continue to flare across Asia and Africa, H5N1 will have vastly more opportunities to adapt to mammals than had been supposed.

Chairul Anwar Nidom of Airlangga University in Surabaya, Indonesia, told journalists last week that he had taken blood samples from 500 stray cats near poultry markets in four areas of Java, including the capital, Jakarta, and one area in Sumatra, all of which have recently had outbreaks of H5N1 in poultry and people.

Of these cats, 20 per cent carried antibodies to H5N1. This does not mean that they were still carrying the virus, only that they had been infected - probably through eating birds that had H5N1. Many other cats that were infected are likely to have died from the resulting illness, so many more than 20 per cent of the original cat populations may have acquired H5N1.

This is a much higher rate of infection than has been found in surveys of apparently healthy birds in Asia. "I am quite taken aback by the results," says Nidom, who also found the virus in Indonesian pigs in 2005. He plans further tests of the samples at the University of Tokyo in February.

The data explicitly contradicts prior statements from the WHO downplaying the role of cats in harboring or spreading the virus, which I wrote about here.  I continue to be fascinated by the extent to which the behavior of the virus in the wild contradicts the expectations and public statements of "officials" in various organizations around the world.  H5N1 is clearly evolving in ways that are both surprising and worrying.

The New Scientist and Science stories both note that two people in Egypt who recently died from H5N1 infections were carrying strains of the virus evidently resistant to  Tamiflu.  It is unclear whether the virus carried the relevant mutations before it infected these patients, or whether it evolved during their illness because they were treated with Tamiflu in the hospital.  Either way, it seems that many people infected with H5N1 are diagnosed after the window in which antivirals are most effective, in part because diagnosis is both difficult and slow.  This phenomenon is described in two articles and a commentary in the 26 November, 2006, issue of The New England Journal of Medicine that report disturbing analyses of human H5N1 outbreaks in Indonesia and Turkey last year.

In a New York Times article about the NEJM papers, Donald Mcneil, reports the following:

Rapid tests on nose and throat swabs failed every time, and in Turkey, so did all follow-up tests known as Elisas. The only tests that consistently worked were polymerase chain reaction tests, or PCRs, which can be done only in advanced laboratories and take several hours.

''It'll be a disaster if we have to use PCRs for everybody,'' said Dr. Anne Moscona, a professor of pediatrics and immunology at Weill Cornell Medical College. ''It just isn't available at a whole lot of places.''

If the A(H5N1) flu mutates into a pandemic strain, rapid tests ''will be really key,'' she said.

What the NYT didn't report is that the patients were infected on average 5 days prior to the appearance of symptoms, outside the window recognized for effective use of antiviral drugs.  Robert Webster and Elena Govorkova have an excellent Perspective piece accompanying the NEJM articles, and they note that in the Indonesian cases, "...Treatment [with oseltamivir] began 5 to 7 days after initial infection.  Such delayed administration of the drug limits its value in decreasing the viral load and might lead to the selection of resistant strains."  It isn't clear from the paper describing the Turkey outbreak when oseltamivir was administered, but those patients did not experience symptoms for an average of 5 days after exposure to the virus, and then received antibiotics for the first 3-7 days of hospitalization before transfer to a unit that treated them for influenza.  In summary, it appears the virus is often being exposed to oseltamivir after the period when the drug is expected to be effective, enhancing the probability of selecting for resistant mutants.

Finally, in a slight change of direction, in the 21 December issue of Nature, John Oxford has a review of a new book on influenza, "Bird Flu: A Virus of Our Own Hatching", by Michael Greger.  You may recall that Oxford is primarily responsible for the hypothesis that the 1918 flu emerged at a British army camp near Etaples, a tale I wrote about a couple of years ago (The Spanish Flu Story).  Oxford notes that:

I am often kicked around by American authors in books about influenza. How dare a Limey suggest that the Spanish influenza A H1N1 virus arose in a gas-infected, pig-ridden and bird-infected army camp of 100,000 people in France in 1916, when the whole world knows it started in Dorothy's home state, Kansas? But I felt less bruised than usual. Perhaps I am getting used to it.

I still find Oxford's version of the origin of the Spanish Flu to be the most compelling, in part because it describes a situation of close contact between animals and people, where those animals were killed and prepared as food by soldiers on a daily basis in conditions not so dissimilar to those in many developing countries where H5N1 is present today.

DNA Vaccines Update and Avian Flu Tidbits

There has been serious progress recently in developing DNA vaccines for pandemic influenza.  First, Vical just announced (again by press release and conference presentation, rather than peer reviewed publication) single dose protection of mice and ferrets against a lethal challenge with H5N1 using a trivalent DNA vaccine.  Ferrets are seen by many as the best model for rapid testing of vaccines destined for use in humans.  According to the press release:

"We are excited by the recent advances in our pandemic flu vaccinedevelopment program," said Vijay B. Samant, President and Chief Executive Officer of Vical. "Earlier this week, we presented data from mouse studies demonstrating the dose-sparing ability of our Vaxfectin(TM) adjuvant when used with conventional flu vaccines. Today we presented data from ferret studies demonstrating the ability to provide complete protection with a single dose of our Vaxfectin(TM)-formulated avian flu DNA vaccine. Our goal is to advance into human testing with this program as quickly as possible, both to provide a potential defense against a pandemic outbreak and to explore the potential for a seasonal flu vaccine using a similar approach."

Mr. Samant will be attending the bio-era H5N1 Executive Round table in Cambridge in a few weeks, along with Dr. David Nabarro, the Senior UN System Coordinator for Avian and Human Influenza.  I'm looking forward to finally meeting these gentlemen in person.

Powdermed is in early human clinical trials for its annual and pandemic flu DNA vaccines in the U.K. and the U.S., and has recently been acquired by Pfizer.  This should provide needed cash for trials, technical development, and perhaps even for building a manufacturing facility for large scale production of their proprietary needle free injection system.  I think it is interesting that a large pharmaceutical company -- a specialty chemicals company, in essence -- has acquired technology that is essentially a chemical vaccine.  I wonder if Pfizer can lend expertise to packaging and DNA synthesis.

Despite progress in the lab and greater funding, there are still significant challenges in getting these vaccines into the clinic.  Here is the DNA Vaccine Development: Practical Regulatory Aspects slide presentation from the NAIAD.  Obviously, lots of work to do there.  And as I have written about previously, it doesn't appear that the FDA is really interested in allowing new technologies to fairly compete, even if they are the best option for rapid manufacture and deployment as countermeasures for pandemic flu.

In other DNA vaccine news, a recent paper in PNAS demonstrated, "Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination."  Kong, et al., showed that, "Immunization with plasmid expression vectors encoding hemagglutinin (HA) elicited potent CD4 and CD8 cellular responses as well as neutralizing antibodies."  Here is more coverage from Effect Measure, which notes that the paper is primarily interesting as a study of the mechanism of DNA immunization in mice against the 1918 virus.

However, if I understand the paper correctly, the authors developed a means to directly correlate the effect of  immunization with antibody production and thereby, "define [the vaccine's] mechanism of action".  This appears to be a significant step forward in understanding how DNA vaccines work.  I interviewed Vijay Samant of Vical by phone a few months ago, and he noted that because animal studies demonstrate complete protection even though traditional measures of immunity do not predict that result, he has a hunch that "tools for measuring immunogenicity for DNA will need to be different than for measuring protein immunogenicity."  Perhaps the results of Kong, et al., point the way to just such a new tool.

An upcoming Nature paper by Micheal Katze, just down the hill here in the UW Medical School, elucidates some of the mechanisms behind the extraordinary lethality of the 1918 virus in mice.  Writing in Nature, Kash, et al., show that:

...In a comprehensive analysis of the global host response induced by the 1918 influenza virus, that mice infected with the reconstructed 1918 influenza virus displayed an increased and accelerated activation of host immune response genes associated with severe pulmonary pathology.  We found that mice infected with a virus containing all eight genes from the pandemic virus showed marked activation of pro-inflammatory and cell-death pathways by 24 h after infection that remained unabated until death on day 5.

In other words, the immune response to infection with the 1918 virus contributed to mortality.  Moreover, "These results indicated a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza requires the use of the fully reconstructed virus."  That is, you have to be able to play with the entire reconstructed bug in order to figure out why it is so deadly.  And this result gives an interesting context to the recent paper of Maines, et al., demonstrating that reassortant viruses of the present H5N1 and lesser strains are not as fearsome as the complete H5N1 genome (which I wrote about a few weeks ago).  This latter observation has been interpreted in the press as evidence that H5N1 is "not set for pandemic", even though H5N1 is demonstrably changing in nature primarily by mutation rather than by swapping genes.  H5N1 is quite deadly, and it may simply be that the particular combination of evolving genes in H5N1 gives it that special something.

Finally, an upcoming paper in J. Virology demonstrates an entirely new antiviral strategy based on peptides that bind to HA proteins in vivo and thereby prevent viral binding to host cells.  "Inhibition of influenza virus infection by a novel antiviral peptide," by Jones, et al., at the University of Wisconsin, appears to still be in pre-press.

In the abstract the authors state:

A 20-amino acid peptide (EB) derived from the signal sequence of fibroblast growth factor-4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo even when administered post-infection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin (HA) protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic.

This is just an initial demonstration, but it is extremely interesting nonetheless.  However, because it is a protein based drug, it risks generating an immune response against the drug itself.  It will have to be administered in a way that preserves function in vivo in humans and doesn't spook the immune system.  The last thing you want to do is generate antibodies against a protein vital for human health.

Yet, precisely because it is a fragment of a human protein, it might mean there is a lower risk of generating that immune response, especially if it can be produced in a way that has all the right post-translational modifications (glycosylation, etc).  Though I wonder about variation in the population: various alleles and SNPs.  What if you are given a version of the peptide that differs in sequence from the one you are carrying around?  Would this generate an immune response against the drug even though it is closely related to something you carry naturally, and if so would those antibodies also pick out your allele?  Definitely the potential for bad juju there.  Another example of where personalized medicine, and having your genome sequence in your file, might be handy.  Alternatively, I suppose you could just use your own sequence for the peptide, and have the thing synthesized in vitro for use as a personalized drug.  Sequence --> DNA synthesis --> in vitro expression --> injection.  Hmmm...you could probably already stuff all that technology in a single box...

However it is used, this advance is probably a very long way from the clinic.  It might go faster if they use the peptide as inspiration for a non-protein drug, which, incidentally, the authors suggest near the end of the paper.  Definitely a high-tech solution, either way, but probably the wave of the future.