A sniper rifle for blood clots

Researchers from Harvard published a report in Science today on the effectiveness of a new nanoparticle based blood clot therapy. They took anti-clotting drugs and packed them in the middle of tiny clumps of nanoparticles. These clumps floated around the blood vessels of mice until they came across a vessel with a blood clot restricting normal flow. Due to the change in the fluid dynamics at these choke points the particles broke apart and delivered the medication directly at the site of clotting.

Critical blood vessel blockage is the leading cause of death worldwide. Currently anti-clotting agents are tricky drugs to dose.  Too little and the clots remain, too much and excessive bleeding occurs. Surgical procedures like stents and bypasses are invasive and result in recovery periods and loss of quality of life. This therapy could get around all those issues.

The drug delivery system worked well in mice, dramatically increasing survival in a fatal pulmonary embolism model system. Hopefully the system holds up to the rigors of more animal model trials and human trials so that someday soon we can treat clotting issues much more efficiently and in a less invasive fashion.  

H5N1 transmission moratorium to expire soon?

Two studies done last year on the creating mammalian transmissible H5N1 influenza caused an uproar over the last 6+ months and led to a self imposed cessation of research into the transmission of highly pathogenic avian influenza in mammals. Originally the research ban was to last 60 days and give the public, government, and research community time to digest the findings and come to a consensus on how to regulate this potentially dangerous research.

Sixty days came and went and the moratorium continued. 120 days passed and still no discussion of ending the ban.  The White House issued a new directive to identify, categorize, and adopt strategies to deal with all current and future dangerous research and the ban went on.  Recently both studies that instigated this controversy have been published and yet the ban continues.
More than 5 months have passed since research into understanding and preparing for these viruses stopped. All the while the threats, both real (natural virus endemic in 6 countries and continuously evolving) and hypothetical (the nebulous terrorists), that the prohibitted research promises to protect against continue to exist.

However, recent chatter suggests the ban may be nearing an end soon. Some participants in the moratorium believe the conditions of ending the ban have been met.  The original research has been published, new improved and more comprehensive guidelines have been established, and governmental funding agencies are now playing a more central role in vetting the research and disseminating the results. Is it time for the research to restart?

Dr. Anthony Fauci

Anthony Fauci, the director of the NIAID, appears to be an advocate of resuming the work sooner, rather than later. Later this next many of the moratorium signers will be gathered in New York for the annual Centers of Excellence for Influenza Research and Surveillance (CEIRS) meeting. Dr. Fauci will be in attendance and plans to address the ban issue while there and hopefully come to a resolution and conclusion.


“I can’t say that the moratorium will officially end then, but we are certainly going to address the topic at that meeting,” says Fauci, “to get some research going on an interim basis.”

Dangerous terrorist? As far as flu is concerned, yes.

Hopefully this discussion will lead to and least a partial end to the ban.  The virus is still present in many parts of the world and is still evolving as you read this. The virus already has some human virus like characteristic and could continue in that directions, especially in places like Egypt and Indonesia. Our best defense against this is research. Let's take the handcuffs off and get back to work.


I will be in attendance at the CEIRS meeting and would be very pleased if the moratorium would end there. Either way I will update what I learn on the subject next month.

Was flu work done 'under the radar'?

One point that Yoshihiro Kawaoka touched uponduring the CEIRS network webinar that I failed to discuss is the idea that the H5 transmission research was done in secret and blindsided the powers that be as has been speculated on in the media.  Let's take a closer look.  

Is that Fouchier or Kawaoka?  See what I did there

  In 2009 The WHO suggested that researchers investigate what it would take to create mammalian adapted H5.  A similar proposal had been issued previously by the NIH in 2006. On top of that, the RO1 grant that paid for Kawaoka's research had 5 specific aims.  The first two specifically proposed the research presented in the controversial paper.  Aim 1) adapt H5N1 from birds to humans. Aim2). induce respiratory transmission.  The methods that his team used to create these viruses have been used previously with other avian influenza viruses to produce similar results (Wan et al., Sorrell et al.).  Finally, all studies involving animal test subjects are reviewed by a panel of university officials, researchers, and outside community members for ethics and welfare. Additionally, the research was reviewed annually by the UW-Madison IBC to ensure no inappropriate research is conducted with potentially dangerous pathogens.

This is not how science happens.

This research was vetted at the local, national, and international level. The studies were not conducted by rogue scientists by misappropriating grant money.  This is not a case of intentionally breaking rules and hoping an apology afterwards is will prevent punishment.  They were not hiding unethical research they wanted to do, but knew others wouldn't understand. ------------>
The university annually inspected it, the NIH approved it and funded it, and the WHO called for it to be done.  To say now that these scientist acted irresponsibly in secret is either revisionist history, or spouting off on something with no understanding of how it works.  But the media would never do that, right?

Where should flu work happen?

With the H5 controversy of the last few months, there has been some talk about moving the mammalian transmissible or even all highly pathogenic flu viruses into BSL4 laboratories.  Here is why it’s a terrible idea.

BSL3, BSL3+, BSL4: What’s the difference?

 

BL3 personal protection

BSL3 is a lab that has certain safety and containment features that go above and beyond a typical molecular biology or chemistry lab (typically BSL2).  BSL3 labs are required to be behind double locked doors. The lab space is required to be maintained under a negative air pressure so that opening doors or unintentional breach of containment causes air to be pulled into the lab as opposed to it  (and what ever is in it) being blown out of the lab.  Additionally, all waste generated is required to be recovered and decontaminated prior to disposal.  Finally, all personnel working in a BSL3 lab are required to wear additional personal protective equipment (PPE). This includes disposable lab coats, multiple disposable shoe covers and multiple gloves, as well as surgical style facemask.

Bl3+ personal protection

BSL3+, other wise known as BSL3 agriculture or BSL3 enhanced, has significant upgrades to the security and safety measures of BSL3.  A BSL3+ is required to have a pass through autoclave so that all solid waste is decontaminated prior to leaving the facility. The researchers body is the only thing that leaves BSL3+ without going through the autoclave (with very few exceptions and only with proper permission and safety protocols).  The scrubs they wear under their PPE are removed in the facility and the researchers are required to shower out in order to leave. PPE is similar to BSL3 but also include a Tyvek coverall and a loose fitting overhead PAPR (powered air purifying respirator) that is equipped with a HEPA filter to scrub the air they breathe. The facility is sealed airtight and all rooms are monitored for pressure changes multiple times a day and the air itself is double HEPA filtered. The entire facility can be decontaminated via gas decon ports from the outside. All security features have built in redundancies and back ups.  Prior to gaining access to a BSL3+ lab, a worker must undergo extensive training and testing and must pass an FBI background check.

A BSL4 lab is the highest level of biosafety and containment possible.  It includes all the elements of a BSL3+ lab with two main differences. Researchers are required to wear a fully sealed suit that air is pumped into from either the outside or a sealed air tank.  These are the moon suits you see in movies like ‘Contagion’ or ‘Outbreak’.  Additionally, the researchers take a chemical decontamination shower to exit the lab. 

BL4 personal protection

Where should it be? 

 

Currently all work done in the US or with US grant money utilizing highly pathogenic influenza is performed under BSL3+ conditions.  All facilities are inspected annually by relevant agencies depending on the pathogens in use. This can include the CDC, NIH, USDA and others. Are these safety features enough for these viruses? BSL3 and BSL4 are reserved for work with pathogens that can cause severe to fatal infections in humans.  The difference between BSL3 and BSL4 is that BSL3 agents have known and effective treatment strategies and BSL4 agents do not.  The only exception being smallpox in BSL4 due to the fact that it has been eliminated from nature.  With this in mind, where should mammalian transmissible H5N1 or all highly pathogenic influenza strains be?  There is no doubt that highly pathogenic influenza is a very deadly pathogen. The ability to become airborne means standard BSL3 conditions are probably not adequate protection.  However the use of PAPRs in BSL3+ are capable of filtering virus out of the air.  The research done with these viruses is very clear.  The drugs and vaccines we currently have are still very effective against these strains making BSL4 inappropriate.  Finally, highly pathogenic influenza is found readily in nature. Humans are exposed to it daily.  All of this leads to one very clear conclusion:  Highly pathogenic avian influenza, mammalian transmissible or not, does not belong in BSL4.  BSL3+ is sufficient protection.  A move to BSL4 would be unnecessarily disastrous, not only to influenza research, but also all current BSL4 pathogens.  A move to BSL4 would kill many flu labs do to a lack of appropriate facilities. Those flu researchers who would still have access to BSL4 facilities would then be jockeying for time and space inside the facilities that already house other research groups.  Retarding research on understanding the most dangerous pathogens is an indescribably terrible idea.  

Even Charles Barkley knows the move is a bad idea.

More on Kawaoka's H5

Kawaoka presented his H5N1 study to the CEIRS network of researchers today via webinar.  Most of this has been covered in other write-ups describing his presentation at the Royal Society earlier in the month, but I will expound on some things that I feel have not been covered in what I have read.  I have a more detailed description of the experiments and results as well as some background on flu here. 

The experiment:

The researchers created an H5 HA that had greatly increased human cell specificity due to three mutations that were randomly added: N158D, N224K and Q226L. They put this HA with the other genes form the 2009 pandemic H1N1 and this virus was able to infect ferrets and showed very weak aerosol transmission (2 of 6 ferrets from 5 to 7 days after infection).  They took a sample of the virus from the animal that was infected via aerosol transmission and re-infected another ferret.  This time the virus transmitted more efficiently (4 of 6 ferrets, 3 to 7 day after infection).   When they sequenced this virus they found one additional mutation, T318I.

They also tested various combinations of the four mutations to dissect how they affect the behavior of the virus. The first three mutations are at positions that are known to affect the species specificity of a virus. The mutations at 224 and 226 affect the shape of the part of the protein that binds to cells. One set of amino acids cause the virus to bind bird cells, the other causes it to bind human cells, kind of like different keys fit in different locks. The mutation at 158 is known to eliminate a glycosylation site. Of all the mutations outlined in the study, this is the only one that has been found in nature in H5 viruses. Many if not most of H5 viruses isolated from birds since 2009 have lost this glycosylation site and every single human H5 lacks it.  According to Dr. Kawaoka’s work, this mutation also serves to stabilize the HA protein. The final mutation also serves this same function.

To me, the importance of this work is not that this is likely going to arise in nature and we should fear it.  The potential of this virus to occur naturally is very low since there would have to be reassortment followed by passage in the right host to build these or similar mutations. The real take away from this paper is how the mutations interact to create a transmissible virus. The two mutations in the receptor binding domain of HA (224, 226) play a major role in switching the host range of the virus, but it also causes the HA to become unstable. This instability prevents this virus from being very infectious and blocks transmission. The mutation at 158 further pushes the virus towards human cell preference but also increases the stability of the protein.  This allows the virus to be more infectious in ferrets and also gives it the ability to transmit through the air (albeit very poorly).  The final mutation at 318 greatly increases the stability of virus and allows the virus to be more infectious and to transmit more efficiently, though still far less infectious and efficient than the 2009 pandemic virus.

With more research on how various mutations affect the performance of a virus, we can start to build an index of mutations and functions.  When surveillance teams isolate new H5 viruses they can identify how new viruses differ from older viruses and can quickly identify how these mutations affect the virus. If officials can identify virus population before they become airborne then vaccine and drug stockpiles can be used more efficiently.  Rather than fully mobilizing our response efforts every time the virus pops up, we can take a more “don’t fire til use see the whites of their eyes” approach and save our ammunition to utilize in the most effective manner. This will ease strains on what experts believe are inadequate stockpiles of drugs and vaccines.

Vs.

Finally we hear about Kawaoka's paper

Last week the NSABB announced that the two controversial papers discussing mammalian transmissible H5N1 authored by Yoshihiro Kawaoka of University of Wisconsin Madison and Ron Fouchier of Erasmus Medical Center.  The bare minimal story in his paper was revealed by Ron back in September at a conference in Malta and again in February at a meeting of the ASM. However, nothing had been revealed about Kawaoka's work until yesterday.

                                       Kawaoka and Fouchier.  credit: M. Enserink
                                                        From: Sciencemag.org
  
Long story short, I am even more confused as to why this work was blacked out in the first place and a little underwhelmed by the results. I guess that is why the board voted unanimously to allow his work to be published.  Basically, Kawaoka took the HA (the protein responsible binding and entering cells; also responsible for species specificity) from H5N1 made a bunch of variations with a bunch of mutations and checked each one for human specificity. He then took a human specific version and plugged it into a virus with other genes from the 2009 pandemic virus.

To recap, this is a virus with 7 fully human flu genes, plus an "avian" gene that has been heavily modified to look human.  When they analyzed the HA gene they found 4 mutations. N158, Q226L, N224K, and T318I.  If you think of a protein as a chain, Q226L means the 226th link in the chain was changed from a Q, glutamine, to L, leucine.  Three of these mutations are fairly well understood.  N158 sits right near a glycosylation site.  Glycosylation sites are where sugar molecules are stuck to the protein and can be thought of kind of like a costume for the virus. Changing the pattern of sugar on the proteins can alter where the virus can bind and how the immune system "sees" and responds to the virus.  The loss of glycosylation right near this mutation has been known to cause a preference switch to human cells.  The mutations at 224 and 226 sit directly in the area that actually binds to the cell surface, the receptor binding site, or RBS.  Making these mutations physically changes the shape of the RBS so different cellular parts fit it. Its like those kid toys with the shaped blocks that fit in different holes. With one set of mutations you have a triangle hole that fits the triangle pegs on a birds cell. Change those to another set and you get a circular hole that won't fit bird pegs anymore, but will fit the human circular pegs.** The last mutation, T318I,  I have no idea about.  It will be interesting to see the follow up experiments they do with that mutation.

                                                  Child's toy or deadly flu mutations?

The virus Kawaoka's team made was able to infect and transmit by respiratory droplets.  Basically when a ferret sneezes it looks like this:

That cloud of white mist is chocked full of virus if the ferret is infected and with the right set of proteins and mutations may be able to infect ferrets (or people) from several feet away. Ferrets that were only in contact with the virus through they air they shared with infected ferrets, but the important thing is that none of the ferrets died. It also seems that these ferrets didn't get as sick as ferrets that had been infected with seasonal or 2009 pandemic viruses.

So we have a highly unnatural, reassorted virus that causes limited disease, but does transmit through the air.  What the hell was the last 4 months about?


**slightly more complicated than triangles and circles.

New dual use federal policy announced

The federal government revised its policy last week regarding potential dual use research. Dual use research is essentially any research that could be used for both good and evil purposes. The research to initiate the new policy was done on highly pathogenic H5N1. Scientists discovered mutations that would allow these deadly viruses to transmit in mammals. This gives researchers, surveillance workers and public health officials a better understanding of what to look for and how these viruses work. However, fears arose that this information could be used by terrorist to make a bio-weapon; thus the dual use designation.

The policy identifies 15 high risk pathogens and toxins and has called for all funding agencies to identify all federally funded research involving these agents within 60 days. Within 90 days the agencies are required to report all instances of research involving the 15 agents that could be considered dual use. Furthermore, the funding agency, institution and lead scientists of studies found to have dual use risks are to create a "risk mitigation plan". This could include modification of methodology, moving research to more secure labs, and alteration of how the research is communicated to the public and scientific community.

The funding agencies "will determine whether it is appropriate to:
request voluntary redaction of the research publications or communications, classify the research, not provide or terminate research funding."

Previously, research done at the NIH and the CDC was reviewed for possible dual use. This was done only for in house studies. The new policy would apply this same standard to all research carried out with NIH or CDC funding. Here is the kicker: these reviews are to be performed for <i>all current<i/> studies as well as future studies. It also appears this will affect research funded by other government agencies such as the USDA and the DOD.

I get why this policy was made. I agree with the vast majority of it. I am not sure how I feel about grandfathering in current research. It doesn't seem fair to drastically change the game half way through.

"Oh you used the CDC grant to conduct your thesis research on HPAI H7N1 and you are about present your 5 years of work to your PhD committee. Sorry, we just classified that so you can't tell anyone about it. Good luck getting a post-doc though."


The 15 agents are:
a) Avian influenza virus (highly pathogenic)
b) Bacillus anthracis
c) Botulinum neurotoxin
d) Burkholderia mallei
e) Burkholderia pseudomallei
f) Ebola virus
g) Foot-and-mouth disease virus
h) Francisella tularensis
i) Marburg virus
j) Reconstructed 1918 Influenza virus
k) Rinderpest virus
l) Toxin-producing strains of Clostridium botulinum
m) Variola major virus
n) Variola minor virus
o) Yersinia pestis

The 7 types of dual use research:
a) Enhances the harmful consequences of the agent or toxin;
b) Disrupts immunity or the effectiveness of an immunization against the agent or toxin without
clinical or agricultural justification;
c) Confers to the agent or toxin resistance to clinically or agriculturally useful prophylactic or
therapeutic interventions against that agent or toxin or facilitates their ability to evade
detection methodologies;
d) Increases the stability, transmissibility, or the ability to disseminate the agent or toxin;
e) Alters the host range or tropism of the agent or toxin;
f) Enhances the susceptibility of a host population to the agent or toxin; or
g) Generates or reconstitutes an eradicated or extinct agent or toxin listed in Section