This season's flu helps inform next season's vaccine

Last year I described the arm's race happening between virus and immune system during an HIV infection: as the immune system starts mounting its defense against the virus, the virus mutates trying to evade the attack. This is what pushes the virus to constantly evolve new strains, not just in HIV, but also in the flu virus, which evolves a new strain roughly every year. The HIV virus evolves within the same host to evade the host's immune response. On the other hand, the flu virus evolves more slowly: contrary to HIV, healthy individuals can clear the flu virus, and in doing so they acquire immunity against future infections from the same virus. This exerts pressure on the flu virus to evolve new strains capable of evading the population acquired immunity.

The process by which viruses constantly evolve new strains in order to evade immune responses is called antigenic drift.

The yearly evolution of the flu virus is closely monitored: surveillance data is collected throughout the flu season and, based on the data, a prediction is made on which strains will be most likely to reappear during the next season -- this step is important for flu vaccine design. The vaccine needs to be available prior to the start of the new flu season. Therefore, researchers have to make an educated guess on what the evolved flu virus will be like in order to make the appropriate vaccine.

"Due to the fast evolution of the influenza virus, the components of the influenza vaccine are changed for many flu seasons. Even though the vaccine is usually redesigned to match closely the newly evolved influenza virus strains, there occasionally has been a suboptimal match between vaccine and virus [1]."

The surveillance data comes from the World Health Organization Global Influenza Surveillance Network (GISN), a network of 136 national influenza centers scattered in 106 different countries. The data focuses on one influenza gene in particular, the hemagglutinin (HA) because the protein it codes seems to drive the antibody response.

The HA protein coats the outer surface of the influenza virus. It enables the virus to recognize and bind target cells. Once bound to the surface of the cell, the virus is engulfed in a sac called endosome. This is a mechanism by which cells engulf extraneous objects and then try to destroy (digest them through enzymes) while inside the endosome. However, the influenza virus uses the endosome to get inside the cell and once there the HA protein undergoes a conformational change (triggered by a drop of pH) and becomes a "hook" that breaks the endosome and frees the virus into the cytoplasm. Without the HA protein the flu virus is unable to bind to the target cell or break the endosome. Therefore, antibodies that bind to the HA protein successfully clear the virus, which is why it is vital for the virus to evolve mutations that enable it to escape those antibodies.

In order to anticipate the next flu strains, researchers need to understand how well the population is responding to the current strains. The flu vaccine usually carries three different strains, selected from the most predominant and geographically spread ones so that the resulting immunity is reactive to a wide range of flu strains. How "different" any two strains are is measured by a quantity called "antigenic distance," which, in layman terms, measures how well current immune responses ("animal antisera raised against the same or related strains, [3]") are able to block those strains. Viruses with a high antigenic distance will be poorly blocked by the current immunological responses and therefore are more likely to diverge from the current flu strains and spread into the following season.

In [3], Smith et al. reconstruct the antigenic map of the influenza A virus starting from 1968. The map retraces the genetic evolution of the virus, showing that strains tend to form clusters that last 2-3 years and then evolve into a new cluster (a new strain that requires a new vaccine). New surveillance techniques are being developed based on this concept of antigenic distance and antigenic maps in order to help inform future vaccine selection.

[1] Pan K, Subieta KC, & Deem MW (2011). A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains. Protein engineering, design & selection : PEDS, 24 (3), 291-9 PMID: 21123189

[2] Cai Z, Zhang T, & Wan XF (2012). Antigenic distance measurements for seasonal influenza vaccine selection. Vaccine, 30 (2), 448-53 PMID: 22063385

[3] Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, & Fouchier RA (2004). Mapping the antigenic and genetic evolution of influenza virus. Science (New York, N.Y.), 305 (5682), 371-6 PMID: 15218094

Antiviral drugs to fight the flu: yes or no?

Disclaimer: I'm not a medical doctor. I cannot recommend taking or not taking a certain drug. However, I am a human being, I've got kids who do get sick from time to time, and I work on viruses. So when I heard that people were battling the unusually nasty flu this year with antiviral drugs, well, I had to do a bit of research.

Antiviral drugs have become increasingly popular after the highly pathogenic avian flu strain emerged. The idea is that in order to be prepared for a possible pandemic, we need to stock up on drugs, enough to treat millions of people.

Let's start with a few facts about viral infections:

A virus is made of genetic material packaged in a tiny shell. Once inside a cell, the virus hijacks the cell's proteins to replicate and create thousands of copies of itself. The new virions bud out of the cell membrane and infect new cells.

However, the infected cell has one more weapon up its sleeve: it "grasps" the virions that are budding out and tries to hold them back. In more scientific terms: there's a molecule on the cell membrane (called sialic acid) that binds to a protein on the surface of the virus (called hemagglutinin). The new virions have to break this bond in order to leave the infected cell and spread the infection. How do they do that? They use an enzyme called neuraminidase: the enzyme, found on the surface of the virus, breaks the bond between hemagglutinin and siliac acid, setting the virions free to spread out and infect new cells.

Neuraminidase inhibitors are antiviral drugs that, as the name suggests, block the neuraminadase enzyme. As a consequence, the virions remain stuck to the cell membrane and thus cannot spread the infection. Supposedly, this leads to a speedier recovery. I'm saying "supposedly" because there is an ongoing debate on whether this is true or not. Here's what I found in the literature.

Jefferson et al., 2009 [1]:

"Neuraminidase inhibitors have modest effectiveness against the symptoms of influenza in otherwise healthy adults. The drugs are effective postexposure against laboratory confirmed influenza, but this is a small component of influenza-like illness, so for this outcome neuraminidase inhibitors are not effective. Neuraminidase inhibitors might be regarded as optional for reducing the symptoms of seasonal influenza. Paucity of good data has undermined previous findings for oseltamivir's prevention of complications from influenza. Independent randomised trials to resolve these uncertainties are needed."

Wang et al., 2012 [2]:

"Oseltamivir and zanamivir appear to have modest benefit in reducing duration of illness in children with influenza. However, our analysis was limited by small sample sizes and an inability to pool data from different studies. In addition, the inclusion of data from published trials only may have resulted in significant publication bias. [...] The clinical efficacy of neuraminidase inhibitors in 'at risk' children is still uncertain. Larger high-quality trials are needed with sufficient power to determine the efficacy of neuraminidase inhibitors in preventing serious complications of influenza (such as pneumonia or hospital admission), particularly in 'at risk' groups."

Jefferson et al. 2012 [3]:

"We found a high risk of publication and reporting biases in the trial programme of oseltamivir. Sub-population analyses of the influenza infected population in the oseltamivir trial programme are not possible because the two arms are non-comparable due to oseltamivir's apparent interference with antibody production. The evidence supports a direct oseltamivir mechanism of action on symptoms but we are unable to draw conclusions about its effect on complications or transmission. We expect full clinical study reports containing study protocol, reporting analysis plan, statistical analysis plan and individual patient data to clarify outstanding issues. These full clinical study reports are at present unavailable to us."

The company that manufactures the brand name drug for oseltamivir responded here, however, according to BMJ, there hasn't been any release of data yet (source).

I have two more cautionary comments.

First: viruses mutate very rapidly and as such, they rapidly find escapes to drugs. An overuse of antiviral drugs may end up selecting drug resistant strains (for example, a flu strain that carries a neuraminadase enzyme that the inhibitor drugs cannot block). I'm not saying that antiviral drugs should not be used. Some life-threatening situations require the use of such drugs (for example, in the case of patients with immunodepression). Other non life-threatening situations don't (plain and simple).

Second: the US Food and Drug Administration recommends the use of antiviral drugs and in fact, last December they expanded the recommendation to children under one year of age (source). However, if you keep browsing the FDA website you find this very interesting Q&A page, where they report some supposed (though worrisome) adverse side effects:

"In the safety review mandated by the BPCA, a number of adverse event reports were identified associated with the use of Tamiflu in children 16 years of age or younger. These adverse event reports were primarily related to unusual neurologic or psychiatric events such as delirium, hallucinations, confusion, abnormal behavior, convulsions, and encephalitis. These events were reported almost entirely in children from Japan who received Tamiflu according to Japanese treatment guidelines (very similar but not identical to U.S. treatment guidelines). The review identified a total of 12 deaths in pediatric patients since Tamiflu's approval. All of the pediatric deaths were reported in Japanese children. In many of these cases, a relationship to Tamiflu was difficult to assess because of the use of other medications, presence of other medical conditions, and/or lack of adequate detail in the reports."

There is no direct evidence that the deaths were linked to the use of the drug. In fact, often it's high risk children that need to take the drug, which means they are likely to have other conditions and take additional medications. If we can't be certain of what one drug alone can do, imagine multiple ones combined. You can read more about the Japan reports here. I also found a reference [4].

Bottom line: drugs are wonderful things. They save lives. Drugs can also mess up with our body chemistry in ways that we don't always understand. The key point is to read, be informed, and use sparingly (as needed, not just as recommended).

NOTE: I strive to make these commentaries as objective as possible. If you feel I've missed some part of the story or you have more references to add to make a rounder point, please let me know in the comments. 

[1] Jefferson, T., Jones, M., Doshi, P., & Del Mar, C. (2009). Neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis BMJ, 339 (dec07 2) DOI: 10.1136/bmj.b5106

[2] Kay Wang, Matthew Shun-Shin, Peter Gill, Rafael Perera, Anthony Harnden (2012). Neuraminidase inhibitors for preventing and treating influenza in children (published trials only) The Cochrane Library DOI: 10.1002/14651858.CD002744.pub4

[3] Tom Jefferson, Mark A Jones, Peter Doshi, Chris B Del Mar, Carl J Heneghan, Rokuro Hama, Matthew J Thompson (2012). Neuraminidase inhibitors for preventing and treating influenza in healthy adults and children The Cochrane Library DOI: 10.1002/14651858.CD008965.pub3

[4] Urushihara, H., Doi, Y., Arai, M., Matsunaga, T., Fujii, Y., Iino, N., Kawamura, T., & Kawakami, K. (2011). Oseltamivir Prescription and Regulatory Actions Vis-à-Vis Abnormal Behavior Risk in Japan: Drug Utilization Study Using a Nationwide Pharmacy Database PLoS ONE, 6 (12) DOI: 10.1371/journal.pone.0028483

Dual-use research of concern

I'm still reading (and very much enjoying) last Friday's Science issue on the flu pandemic. In [1], Anthony Fauci and Francis Collins summarize very well why the flu presents a potential threat:

"Influenza viruses have animal reservoirs, especially in birds and pigs. They can undergo extensive genetic changes and even jump species, sometimes resulting in a virus to which humans may be highly vulnerable."

Over the last hundred years, this happened four times: in 1918 (the Spanish flu), in 1957 (the Asian flu), in 1968 (Hong Kong flu), and, the last time, in 2009 with the H1N1 pandemic (swine flu). H5N1 has not initiated a pandemic because it is rarely passed between humans and it infects only through direct contact with infected birds. So far there have been about 600 total cases, of which nearly 60% have resulted in deaths (though this last number is likely to be inflated as often not all non-fatal cases are reported to hospitals). Given these statistics, if the virus were to spread more easily (for example through sneezing or coughing), this would entirely change the threat level, hence the concern when two labs independently announced that they had found an H5N1 mutant able to spread through aerosol in ferrets.

"One of the goals of pandemic influenza research is to recognize and anticipate how viruses are evolving in the wild toward a phenotype that is dangerous to humans, thereby staying one step ahead of potential pandemics."

That's why studies like the ones conducted on ferrets are so important. Yes, the virus was genetically engineered in a lab, but once you have it, you can address the following questions: how likely are those mutations to appear spontaneously? Can we use the virus to produce a vaccine? Are the current antiviral drugs successful in containing the infection or is there a need to develop new drugs?

"However, whenever one deliberately manipulates a virus or a microbe, it is always possible, at least theoretically, that the research results could be used by bioterrorists to intentionally cause harm, or that an accidental release of a pathogen from a laboratory could inadvertently cause harm."

Any research that presents such dual potential of benefit and risk/threat is referred as DURC, which stands for dual-use research of concern. The controversy around the two H5N1 papers clearly proves that we need better ways to assess and regulate DURC research, as well as mitigating the risks while highlighting the benefits.

"There is still no consensus on how to practically define DURC; whether it is feasible to identify and regulate DURC experiments; how to address risks associated with DURC; and how to balance this risk with the necessity of fostering life sciences research for public health and biodefense [2]."

It is a necessity for scientists to be open about their research. Research is rarely conducted in isolation. Open discussion is what fuels great ideas. A DURC policy that requires new protocols and manuscript redactions threatens this openness. Furthermore, it isn't feasible to retroactively restrict scientific information since most scientists are required to write reports describing their current research, as well as give talks and presentations at official meetings. When the NSABB made its recommendation not to publish the two H5N1 studies last year, it was probably already too late. Many people in the scientific community had already heard sufficient details, even prior to their publication.

What are your thoughts on the matter?

[1] Anthony S. Fauci,, & Francis S. Collins (2012). Benefits and Risks of Influenza Research: Lessons Learned
Science, 336 (6088), 1522-1523

[2] Carrie D. Wolinetz (2012). Implementing the New U.S. Dual-Use Policy
Science, 336 (6088), 1525-1527

Flu pandemics: are we prepared?

The first avian flu controversial paper appeared in Nature last April, and the second one just came out in the last issue of Science. For the occasion, Science also published a series of essays from various experts in the field discussing the issues at stake and what we learned. The most fundamental question is: in case of a flu pandemic, are we prepared?

Rino Rappuoli and Philip Dormitzer discuss what can be done in the event of a flu pandemic, basing their observations on the lessons learned from the 2009 H1N1 (the so called "swine flu") pandemic [1]. In 2009, H1N1 vaccines were successful in reversing the rise of flu cases. However, they became available only after the pandemic had started (8 months after the first case had been reported) and only in wealthy countries.

The authors present four possible preventive approaches.

(1) Pre-pandemic vaccination: in 2009, people exposed by either vaccination or infection to the H1 strain, were able to sustain the H1N1 infection. If a vaccine induces a strong antibody response, those antibodies will work also against distantly related strains, indicating that vaccination against the H5 strain may be able to prevent a possible H5N1 pandemic.

"Because H5N1 infection of humans is so rare, the population has not been primed by previous exposures. This lack of prior exposure makes the population more vulnerable to severe disease caused by highly virulent H5N1 strains and makes immunization more difficult, necessitating the use of adjuvants, higher antigen content, and two doses to elicit sufficient antibody titers."

(2) New cell culture technologies: currently, most vaccines are grown in cell cultures, which is dependable on egg supplies and lengthens the production time. New cell culture technologies would increase the manufacturing capacity of the vaccine, which is needed because mathematical models show that "the proportion of the population that must be immunized for effective immunity is likely to be greater than 80%."

(3) Extended Program on Immunization (EPI): currently only rich countries have broad vaccination policies and not many recommend routine flu immunization. Initiated by the World Health Organization, the EPI program sets goals and a standardized vaccination schedule for all children world-wide. Currently, flu vaccinations are not part of the EPI program, but if they were introduced within the program this would expand the availability of the vaccines beyond the rich countries.

(4) Synthetic seed generation: a viral strain is needed to make a vaccine and cloning these strains takes time. Rappuoli and Dormitzer propose as an alternative synthesizing the genes instead to generate synthetic virus. Novartis, the multinational pharmaceutical both Rappuoli and Dormitzer work for, has an ongoing project to use synthetic genomics that would reduce vaccine production time by up to two months.

"In conclusion, while we wait for the development of a universal influenza vaccine, we have practical options that we could implement today to reduce the risk of mass global mortality from the next influenza pandemic."

[1] Rappuoli1, R, & Dormitzer, P. (2012). Influenza: Options to Improve Pandemic Preparation Science, 336 (6088), 1531-1533 DOI: 10.1126/science.1221466

Avian flu papers will be published!

Apologies if you've heard about this already, I was away last week and I'm slowly catching up.

The members of the National Science Advisory Board for Biosecurity (NSABB) finally gave the green light for the two papers on the avian flu virus to be published in full in Science and Nature respectively. I originally discussed the issue in this post, and you can read about the update here. Basically, the two papers independently showed that an artificially mutated strain of H5N1 (the so-called avian flu, a highly pathogenic flu strain, under normal conditions transmissible only through contaminated fluids) could be transmitted through aerosol (not just fluids) in ferrets. Back in December, the NSABB had recommended not to publish the papers in full, and in particular to redact the data on the mutations that conferred the new transmission route to the virus.

Security issues aside, which I have already discussed, the part that struck me about the Science news story [1] is this:

"The original papers were typical Science and Nature papers: very brief, short on detailed discussion, little to no information on biosafety/biosecurity/mitigation, and perhaps even a little sensational," says NSABB member Lynn Enquist, a molecular biologist at Princeton University. Fouchier's original paper, in particular, was somewhat misleading, several NSABB members told Science. [. . .] Fouchier agrees that his original 2500- word Science manuscript was "not as clear and as explicit as it could have been if we had been given another couple of hundred words."

It's an issue I am very sensitive to because even though I understand high-profile journals like Science need to keep a word limit, we (authors) often struggle to meet this limit and have to sacrifice clarity in order to keep the report concise. Invariably, the reviewers come back with confused comments. I don't have a solution to this, but it is a problem. In this particular case, the solution was to allow Fouchier and colleagues extra words to present the data.

The other statement I'd like to highlight is one NSABB member Michael Imperiale of the University of Michigan Medical School in Ann Arbor made, also reported in [1]:

"What it came down to for me ... [is that there] might be a risk to not publishing."

Something I completely agree with. Sharing the data will motivate more funding into this type of research and more surveillance.

[1] Cohen, J., & Malakoff, D. (2012). On Second Thought, Flu Papers Get Go-Ahead Science, 336 (6077), 19-20 DOI: 10.1126/science.336.6077.19

Is epigenetics new? Not to a "smart" influenza virus!

I was browsing the latest papers on Science when I read:

"By mimicking epigenetic regulation in human cells, one flu strain suppresses the expression of antiviral genes [1]."

Wow. Epigenetics and viruses? I had to read that paper!

I've discussed many times how gene expression in cells can be altered through epigenetic changes. The figure below, also taken from [1], shows one of the most common mechanisms by which cells alter gene expression: inside the nucleus, DNA is wound around proteins called histones. The addition of a methyl group (shown in blue in panel b), can alter the transcription of the gene.

In the figure, H3 represents the histone tail. This tail has two amino acids, lysine and arginine, which can be modified in a way that alters the interaction between DNA and the histones. These cause changes in the topology of the chromatin (the way the DNA is packaged inside the nucleus), allowing for certain regions rather than others to be accessed for transcription. If I just gave you a headache, think of chromatin as a tight yarn and you want to poke your finger inside to reach certain threads. The histone tail is like a "lever" that you can use to gain or lose access to the inner parts of the yarn. That's how the cell activates or deactivates genes.

"The ability of histone tails to guide gene function indicates the possibility of targeted control of gene expression by artificial or naturally occurring molecules that can structurally and/or functionally mimic the histone tail [2]."

The influenza A H3N2 subtype has a protein, NS1, that is not vital to viral reproduction but is known to suppress the host's response to the viral infection. In fact, without NS1, the viral infection is significantly mitigated. In a recent Nature paper, Marazzi et al. [2] found that NS1 carries a sequence that resembles the histone H3 tail. This mimicry supports viral infection by halting the transcription of genes essential to counteract the infection.

"We have shown that H3N2 influenza A virus interferes with host gene expression by exploiting the very basic principles of the epigenetic control of gene regulation. By mimicking the histone H3K4 sequence, which has a key role in positive regulation of gene transcription, the influenza virus gains access to histone-interacting transcriptional regulators that govern inducible antiviral gene expression."

Of course, these findings are very intriguing and they raise the question of whether this mechanism is novel to the H3N2 strain or not, and, also, what would happen if a particularly virulent strain like the avian flu would suddenly develop this mechanism as well. At the same time, this opens up new research on ways to attenuate viral infections by targeting the NS1 protein.

[1] Krasnoselsky, A., & Katze, M. (2012). Virology: Influenza's tale of tails Nature DOI: 10.1038/nature11034

[2] Marazzi, I., Ho, J., Kim, J., Manicassamy, B., Dewell, S., Albrecht, R., Seibert, C., Schaefer, U., Jeffrey, K., Prinjha, R., Lee, K., García-Sastre, A., Roeder, R., & Tarakhovsky, A. (2012). Suppression of the antiviral response by an influenza histone mimic Nature, 483 (7390), 428-433 DOI: 10.1038/nature10892

Avian influenza, ferrets, and bioterrorism: fear versus science

I learned about this last week, when Science published a short article on how the National Science Advisory Board for Biosecurity had recommended two research groups NOT to publish details on how avian influenza strains were modified in order to make them transmissible through aerosol in ferrets.

You can read that story here.

The first thing that struck me was: is this censorship? Because for as long as I've been a scientist I've known that the great bulk of scientific progress is made through the free exchange of ideas and results. The very core of scientific validation is in the reproducibility of an experiment, and you can't reproduce an experiment unless who conducted it shares the details.

Why then the recommendation?

The World Health Organization currently lists the case fatality of avian influenza (H5) somewhere between 50% and 80%. This is the percentage of all cases that report in a hospital and have been confirmed through labwork. Currently, it is transmissible through fluids by coming in contact with infected birds. The two studies under the radar here, by Ron Fouchier at Erasmus Medical Center in Rotterdam and by Yoshihiro Kawaoka at the University of Wisconsin, have been submitted but not yet published to Science and Nature respectively. Though different, they both prove that it takes a relatively small number of mutations for the virus to become transmissible through aerosol in ferrets.

Why the fear? With a fatality rate anywhere above 50%, if you can make the virus transmissible through aerosol, you've got a deadly weapon. But is it so obvious one can make it?

First, ferrets are not humans and currently we have no way to predict whether what has been observed in ferrets is likely to happen in humans. For example, there are many strains of avian influenza, and they all have been circulating in birds for many decades. However, of all these strains, only three (H1, H2, and H3) have been able to circulate in humans. There is a natural bottleneck in the way a virus is able to adapt from one organism to another.

One may object we don't know for sure, so, theoretically, it could be possible. But in that case, is censorship the answer? I honestly don't think so and I was quite happy to find a PNAS paper [1] in complete agreement with my thoughts:

"Why Is it Important to Have the Full Data Published? With respect to the specific papers by Fouchier and Kawaoka, it would be important for other scientists to replicate portions of these works to test new vaccines/therapeutic agents and for continued studies on the molecular aspects of influenza transmission, a topic that is extremely important yet relatively poorly understood."

And, most importantly:

"It would be very difficult for a bioterrorist to come up with a human virus strain that is transmissible and still highly virulent. Under natural conditions, however, there is virtually unlimited allowance for generation of capable viruses, the opportunities for infection of humans are plentiful, and the evolutionary pressures of selection are great. If anyone could do it, Nature could."

And that's exactly why we need to be prepared. And the way we are prepared is by sharing results and having multiple groups worldwide brainstorm and join forces to find a vaccine.

What do you guys think?

Palese, P., & Wang, T. (2012). H5N1 influenza viruses: Facts, not fear Proceedings of the National Academy of Sciences, 109 (7), 2211-2213 DOI: 10.1073/pnas.1121297109