When a Teardrop Can Kill: The Deadly Ebola Virus

Posted on 26 March, 2015 by Neil

March 22nd marked a grim first anniversary: what we now know is the deadliest Ebola outbreak to date was officially announced by the WHO in Guinea. Since then, news reports have featured the dramatic and worrying outbreak of Ebola virus infections in West Africa. In our blog post this week, ICaMB’s Dr Anjam Khan reviews this deadly but scientifically fascinating killer and reflects on his recent experiences discussing the virus in the media.

By Dr Anjam Khan

Prophecies of Science Fiction

As a school kid I was fascinated by science fiction and the visionary predictions made about future technology by the fathers of this genre, Jules Verne and H. G. Wells. Science fiction writers have also forewarned of global catastrophes ranging from those caused by climate change, to the annihilation of the human race by a virus concocted in the lab of a mad scientist! A book called “The Andromeda Strain”, by the best-selling author Michael Crichton, captivated me. The story was set in Arizona. It followed a team of scientists in a secret high containment lab trying to control the apocalyptic spread of a deadly extra-terrestrial microbe. The infection rapidly caused lethal blood clotting in its victims, or “disseminated intravascular coagulation” to us nerds! The crystalline alien microbe “mutated” at an extraordinarily high frequency, almost instantaneously acquiring new biochemical skills such as the ability to digest through plastics and rubber seals. These are attributes synthetic biologists dream of constructing today in “environmentally friendly” bugs to degrade plastics!

The Devastating Realities of Ebola The deadly Ebola virus outbreak is not from the realms of science fiction but written by nature. This outbreak has shocked the world not only because of the high mortality rates, but due to the horrific disease it causes. The virus has quickly spread and brought the three West African countries, Guinea, Sierra Leone, and Liberia, to the their knees, and to the brink of economic collapse.

Map of West Africa with Guinea, Sierra Leone, and Liberia highlighted. Image from copyright free – morguefile.com

Reports of the first cases of Ebola in Europe and the US brought panic and fear to those who previously thought this deadly plague was confined only to West Africa. Undeniably, governments and healthcare organizations were all taken by surprise, and were ill prepared to rapidly deal with the disease or indeed its global threat. Why had Ebola taken the world by surprise? The warning signs have been there for almost four decades! The first case of the disease was described in 1976 in Zaire (now the Democratic Republic of Congo), near the Ebola River from where the virus gets its name. With the present outbreak there have been over 23,000 reported cases with approximately 10,000 deaths. The human toll in fatalities has been almost 10 fold greater in the current outbreak than all the previous outbreaks put together!

Politics of Disease Control and Media Engagement The world hesitated in responding to the emerging Ebola crisis, naively hoping the disease would burn itself out. This did not happen and the number of newly infected individuals increased week on week. Some politicians and members of the media fuelled the fears of the masses. Our government hurriedly announced it was establishing four NHS hospitals, including the Newcastle Royal Victoria Infirmary, as Ebola centres with highly trained staff and fully equipped facilities to deal with Ebola cases. Furthermore, compulsory thermal scanning of travelers arriving in airports from selected destinations was introduced, in a bid to spot anyone with a fever and potentially infected with Ebola. There are, however, many other infections and health conditions which can also induce severe fever.

Thermal cameras to detect passengers with high fevers were recommended in airports for screening for potential Ebola infected individuals. Photo Credit: cdc.gov

Hence, in reality this screening approach was only of limited value in identifying Ebola infected travellers, and perhaps served more as a placebo to allay the fears of the public. For example, Thomas Eric Duncan was thermally screened upon arrival in Texas, and he later developed full-blown Ebola and sadly died of the disease in Dallas. Two nurses treating him also became infected.

The national and local news media wanted to find out more about Ebola and the control measures being put in place. With a background in infectious diseases, I was asked to provide insight into the virus and the disease it causes. Normally I would run a mile from being in the public eye. But given some gentle encouragement, decided on this occasion to raise my head above the comfort zone of the academic parapet, and try to provide sensible and factually correct advice on the virus and the disease. Following my first interview with a local newspaper the requests for interviews increased, and within a few weeks I had given three television, four newspaper articles (Northern Eco, Chronicle, The Week), and a radio interview! This included one live TV interview for BBC News 24 via a satellite link, which was petrifying! It was challenging not only because it was being transmitted live, but due to the technical arrangement – but that’s a blog for another day! Speaking to the media was a brand new experience for me. It was certainly a steep learning curve and I realised the importance of “sound-bites”: you can be interviewed for 20 minutes but the editor may only select two 15 to 20 second excerpts for airing. Furthermore you quickly realise you have no editorial control of the content or indeed the context of your quotes!

Pirates of the Immune System

A typical fruit bat: Photocredit: Wikipedia.org

The first Ebola outbreak in 1976 was identified in Central Africa, and it has now spread to Western Africa. This distance at first glance may not sound very far, but to put this into perspective, it is comparable to the distance between Newcastle (UK) and Quebec (Canada) or between Newcastle and Cairo (Egypt)! A key factor contributing to this spread could be the fruit bats, which harbor the virus, flying and migrating to new habitats. For reasons unknown as yet, some fortunate individuals can recover supported by simple hydration therapy. These survivors provide a hugely invaluable resource of biological information, and will undoubtedly provide important insights into understanding the immunological correlates of protection or the genetic basis of resistance to Ebola. But sadly for the vast majority of infected individuals, the disease is devastating. The cunning virus “pirates” the immune system and incapacitates the anti-viral machinery of the immune cells by blocking the “interferon alarm” from sounding in target cells. The virus then triggers a “cytokine storm” triggering the release of potent inflammatory molecules into the circulatory system that wreak havoc throughout the body, and causing organs such as the liver and kidneys to fail. Blisters of blood erupt below the skin. During these final stages of haemorrhagic fever clotting factors can become depleted (compare to the Andromeda Strain!), and blood vessels start to leak profusely, heavily tainting vomit and diarrhoea with blood. Shockingly, infected individuals literally bleed to death through every bodily orifice from their eyes to their ears! . It is during this final phase the virus is most infective due to the very high concentration of virus particles in the blood and body fluids. Undiagnosed patients can inadvertently spread the killer disease to family and healthcare workers. There is nothing more heart breaking than watching a distraught infected mother not being able to hug and console a crying child for fear of spreading the disease. This is an unforgiving virus and literally a teardrop can kill!

The Achilles’ Heel of a Giant

As we learn more of the biology of the Ebola virus, scientists are uncovering ways to prevent or tame infections. The fatal power of this giant-sized virus is largely attributed to a single protein, known as the “spike-protein” and is the virus’s “magic wand”. Located on its surface, this protein is crucial for infecting cells, as well as manipulating the immune system. The shrewd virus cloaks and masks key domains of this protein using sugars, allowing it to evade the patrolling cells at the frontline of our immune systems. This spike-protein could also prove to be the virus’s Achilles’ heel, and insights into its structure will enable scientists to design new vaccines and drugs to target and inactivate vulnerable uncloaked regions in this essential protein.

There are now prototype vaccines under development and undergoing fast-tracked human trials. A therapeutic cocktail of genetically engineered monoclonal antibodies known as ZMapp have been successfully used to neutralize the virus, providing passive immunity and protection against disease. The Ebola outbreak has also motivated me to return to my background in vaccine discovery and help contribute towards the development of vaccine against Ebola. To this end I have established a collaboration with my good friend Pietro Mastroeni (Cambridge) and Gary Kobinger (Canada). Gary is a pioneer in the field of viral haemorrhagic viruses, and senior author of the recent article in Nature describing the reversion and protective effects of the ZMapp antibody cocktail against Ebola. Our strategy will be to use synthetic biology to engineer an oral delivery platform for the vaccine, obviating the need for needles and syringes, or the requirement for refrigeration.

Global Health Security

Global health security www.cdcfoundation.org

Ebola has been considered a rare disease and consequently very little money has been invested in research or the development of therapeutics or vaccines to protect against disease. Big pharma have certainly steered clear for commercial reasons. The world governments, pharma, and international healthcare agencies need to co-operate and forge an alliance in readiness to prevent this and future outbreaks of infectious diseases from happening again. The international community has been slow in learning from the re-emergence of polio, cholera, or from the recent outbreaks caused by pathogens jumping from animals to humans. These include bird or swine flu (H7N9; H1N1), and the severe acute respiratory syndrome coronaviruses SARS and MERS. To keep the world safe from threats of infectious diseases, a major input of finance and resources from the national and international communities is required to provide essential support for research in microbiology and immunology, and establishing the necessary medical and management infrastructures essential in dealing with future episodes of Ebola. The present Ebola outbreak fortunately is now being brought under control. However in recent days there has been a British healthcare worker who has become infected in Sierra Leone, and her close contacts have also been flown back for treatment and monitoring in London and Newcastle. Fortunately, the Ebola outbreak will not become the global pandemic we all feared, and the UN predicts the outbreak will be over by the summer of 2015. However, big questions remain: Where does the virus go to in between these sporadic and unpredictable outbreaks? Does the virus fester away increasing in numbers in populations of fruit bats, waiting for the opportunity to jump across into primates? This is my great fear! The world should treat the present tidal wave of Ebola as a wake-up call. I hope I am wrong, but suspect complacency will prevail as Ebola begins to fade in our memories over the coming months. There is an apt quote from William Arthur Ward “The pessimist complains about the wind; the optimist expects it to change; the realist adjusts the sails”. The international community must become “realists”, and adjust their responses to act now and fulfill all their promises of funding and resources! Otherwise, this could provide a tragic opportunity for a microbiological tsunami to hit the shores of every country!

What did the Black Death ever do for us? The curious route to an Industrial Biotechnology Catalyst Grant

Posted on 12 March, 2015 by Neil

UK Government Minister Vince Cable recently announced the results of the first round in the BBSRC-supported Industrial Biotechnology Catalyst scheme where £20 M was distributed across 23 projects. Here, ICaMB’s Jeremy Lakey describes the curious scientific route that led from researching Yersinia pestis, the bacteria responsible for the bubonic plague, to a potential biotechnology breakthrough.

By Professor Jeremy Lakey

The project that I put together with Neil Perkins (ICaMB), Dave Fulton (Chemistry), Matt German (Dentistry) and Nick Reynolds (Dermatology), called (rather snappily I think) Manufacture of complex protein polymers for industry and medicine, is one of the recently announced Industrial Biotechnology Catalyst awards. It’s a £2.4 million research programme with a BBSRC 80% contribution of £1.8 million and quite honestly a year ago I’d never imagined having this amazing five years funding to realise this project that has nagged at me for at least the last five years.

The early years

My interest in the Caf1 protein first arose from a project with the Defence Science and Technology Laboratory (DSTL) on vaccines against possible bioweapons such as anthrax and plague. Other labs had shown Caf1 to be a chaperone usher (CU) family protein, secreted through the outer membrane of the plague bacterium Y. pestis as non-covalently linked polymers. However, most members of this family were visible under the electron microscope (EM) and had a defined structure. We tried in vain to see it under the EM for a couple of years and had given up but, as luck would have it, the world authority on EM observation of proteins using negative stain, Robin Harris, had retired from his job in Germany and moved to Hexham a town in the Tyne Valley near Newcastle. He agreed to have a look with my student at the time, Andrei Soliakov. By adding very low concentrations of protein and using his magic staining recipe, they got amazing images on the first day (see figure). I was on the other end of a country modem at my brother’s farm and so for me the images unfolded slowly down the screen. Robin, Andrei and I (much later in the day), were thus the first folk ever to see one of the key proteins of the Black Death which, between 1346 and 1353, killed up to half of the population of Europe. It was one of those sobering moments in life.

The Caf1 polymer as seen using an electron microscope (top). The structure (middle) resembles a line of circus elephants (bottom).

We also imaged these amazing molecules on the vaccine adjuvant, they looked like Christmas paper chains hanging off the sides of the adjuvant particles. No one had seen Caf1 or proteins on adjuvants before, even though most of us have been vaccinated using this material. I was swept away by the sheer coolness of the data we had and submitted a paper to Nature, then Science, then somewhere else and somewhere else after that but it soon appeared no one was quite as amazed as I was. This remains true to this day with the eventual paper in Vaccine (Ref 1 below) still only having 2 non self- citations; something I still fail to understand.

It’s love actually

However I was seriously smitten by this molecule and hardly cared. This infatuation was made worse one day when, in a seminar, somebody showed the structure of the extracellular matrix protein fibronectin or at least the domains around the integrin binding sites where the well-known RGDS sequence motif is situated. These protein domains looked just like Caf1 because they were both immunoglobulin-like domains.

Different immunoglobulin-like protein topologies (from Pyburn et al, PLoS Pathog. 2011)

Of course, I should have seen this before as I go on and on and on about protein families in my second year undergraduate lectures. About 70% of cell surface and extracellular matrix proteins are built at least partly from this simple domain structure. Like other superfolds such as the TIM barrel and globin these are protein structures with no sequence homology that are found in large numbers across biology and partly explain why there seems to be a limit to the number of different protein folds (Ref 2). But that is a story for another day.

The immunoglobulin-like fold is found in immunoglobulins (surprise, surprise), MHC, fibronectin, many surface receptors such as EGF receptors etc. etc. It’s also found in the largest protein known, Titin, which stops our muscles over extending when they are stretched. Now it turns up in this Caf1, which is beginning to nag me constantly with the thought that surely I could make things from this amazing polymer. So, off I go to the BBSRC with Mark Birch as co-investigator. With Liz Mitchell we had just shown that we could induce bone formation by growing osteoblasts on surfaces coated with engineered small proteins containing sequences from Bone morphogenic protein 2 and osteopontin (Ref 3). I said I could modify Caf1 to do the same and we could form it into hydrogels and grow 3D bone amongst other tissues – what could possibly go wrong. Funnily, the BBSRC did not agree and forbade any similar grant from darkening their doors for seven generations or something that sounded like that.

Try, try and try again

The project, such as it was, retreated to lick its wounds. At least my lack of citations meant that no one else in the world was likely to be working on it but neither it seemed was anyone interested in funding it. Salvation came in two forms. One was the MRC Industrial Collaboration studentship scheme, which enabled us to apply for money with Orla Protein Technologies Ltd, a spinout company I had co founded with Dale Athey in 2002. Orla sells engineered protein surface coatings such as those we used in the BMP/Osteopontin paper and very good they are too, try them yourselves at www.orlaproteins.com!

The second bit of luck was that Ana Roque applied for the studentship. She is an amazingly tenacious and hardworking scientist who single handedly made the Caf1 project work. First she tackled the ridiculously large Caf1 plasmid and won. We then had a manageable system that could produce mutant Caf1 in good amounts.

Immunoelectron microscopy of Y. pestis showing macrophage resistant hydrogel capsule From Du Y et al. Infect. Immun. 2002;70:1453-1460

She then showed that cells were not keen on growing on Caf1 surfaces. Not good. What turned it around was that by simply adding an RGDS motif we could make Caf1 act like fibronectin with cells sticking to it like last night’s pigs trotters. Caf1 makes a macrophage resistant hydrogel capsule around the Y. pestis cell that prevents interaction with cells (see figure). Ana had thus shown that it keeps its non-adhesive properties in vitro… whoo-hoo!. Thus, Caf1 is a non-stick, tough, flexible, polymer – all things very difficult to build into a protein by design. It can be genetically modified to imitate at least one Extra Cellular Matrix (ECM) protein and physically resembles many more. Ana then showed that we could cross-link it into hydrogels and her pièce de résistance was to show that we could make mixed polymers by expressing differently modified domains in the same cell and letting them mix randomly in the emerging polymer. This was published in Advanced Materials (Ref 4), which will do nicely as one of my REF papers although I am not waiting for the citations to roll in any time soon.

Ana returned to Portugal and the Caf1 project was halted again. We got some short term funding to pay Helen Waller’s salary to make more protein and more mutants.

In November 2013 the Technology Strategy Board (TSB – now Innovate UK) announced the Industrial Biotechnology Catalyst with £20M in the first round. Although I have been involved with the bioprocessing industry for many years I did not think the Catalyst was for me, as it seemed to be either biopharmaceuticals or bioenergy. However, I read the outline for the feasibility awards and it seemed to fit Caf1. I was thinking of a post doc for three years etc but the award size started at £2M, so some imagination was required. This led to the current project. Protein engineering to design and produce new polymers will be done in my laboratory. This will be done by Helen Waller and a new post-doc. David Fulton will employ another post-doc to make our cross-linked hydrogels smart and responsive to a range of stimuli like temperature, light and pH. Matt German will use impressive kit like his Atomic Force Microscope (AFM) to measure the material properties of the gels whilst Nick Reynolds will continue our work on wound care. Last, but not least, ICaMB blog’s very own Neil Perkins will work with another new post doc to understand how cells respond to and remodel the different materials. Neil’s suggestion that his qualification was that he was the first person I met that day is unfounded, the truth is that his title of Prof Gene Expression and Signalling fitted the number of words left in that section of the form (I maintain that the previous 2 people Jeremy had bumped into that day had turned him down – Neil).

To boldly go…

So, we have five years to turn Caf1 into a range of 3D cell culture products. By exploiting bacterial production we hope to reduce costs and provide bulk materials at affordable prices for a series of applications. These will include research tools for cell culture, better culture conditions for industrial processes, tissue and regenerative engineering materials and many more. I have produced products in the past and it is very different to our normal hypothesis driven discovery research. In discovering things about nature you generally have observed something and then set out to find out why it’s like that or how it works. In product invention you never know if it will ever work because, by definition, no one knows (and that is particularly true of protein engineering!). The Industrial Biotechnology Catalyst money is aimed at expanding the UK biotechnology industry and, in taking the money, we must be aware that quite justifiably the tax payer wants to see a return on the investment. Ultimately we hope we can create jobs in a new area based upon this project. What’s more, collaborating across disciplines to invent amazing new materials and use them to cure disease is a great way to spend ones working life.

[1] Soliakov A, Harris JR, Watkinson A, Lakey JH. The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states. Vaccine. 2010;28:5746-54.

[2] Orengo CA, Jones DT, Thornton JM. Protein superfamilies and domain superfolds. Nature. 1994;372:631-4.

[3] Mitchell EA, Chaffey BT, McCaskie AW, Lakey JH, Birch MA. Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro. BMC Biology. 2010;8:57.

[4] Roque AI, Soliakov A, Birch MA, Philips SR, Shah DS, Lakey JH. Reversible Non-Stick Behaviour of a Bacterial Protein Polymer Provides a Tuneable Molecular Mimic for Cell and Tissue Engineering. Advanced Materials. 2014;26:2704-9.

Happy Birthday PAN!C

Posted on 5 March, 2015 by Kevin

By Beth Lawry

ICaMB’s postgraduate student association (PAN!C) celebrated it’s 2^nd birthday in February. Thank you to all 40+ of the ICaMB postgraduate students who came and enjoyed our night of celebrations!

Yep, if you’re an ICaMB postgraduate student, I’m the one who clogs up your inbox with emails about yet another PAN!C activity. The past few years, since Claire Whitworth and Kerrie Brusby began the ICaMB postgraduate association, have been an amazing whirlwind. To celebrate PAN!C’s birthday I thought I’d share my highlights with you.

PAN!C CV Workshop, June 2014

In February 2013, PAN!C applied for and won a University Innovation Grant, and were invited to celebrate with the Vice Chancellor. These funds have enabled PAN!C to host numerous events, both academic and social, for ICaMB’s PhD student community. In November 2013 we hosted our careers symposium. Of course, we’ve all been given the usual, generic career options for people with PhDs, but that wasn’t enough for PAN!C. We wanted our students to have the opportunity to hear about careers they perhaps had not thought of, and provide the chance to meet professionals in these areas…. We even had an interesting insight into running a pole dancing business! In 2014, PAN!C hosted a CV workshop, with experts providing information on how to improve and tailor your CV to a variety of sectors. We had 12 speakers, and over 60 students attended the day.

PAN!C Climbing, February 2014

Both the Careers Symposium and CV workshop received extremely positive feedback, from students and speakers alike, with 100% of responders stating they’d recommend the workshops. It’s worth noting that, while organising these events, I made contacts with people from industry, teaching, law, business and recruitment, all of whom I’m still in touch with. PAN!C has provided me with a fantastic opportunity to network with my peers, and gain skills in team work, grant applications, leadership and communication.

PAN!C are crowned FMS quiz champions, May 2014

On the social side, PAN!C has always strived to bring together the many groups within ICaMB. Together we’ve conquered our fears and climbed to dizzying heights at the Newcastle Climbing Centre, skated on ice at the Centre for Life in a somewhat Bambi fashion, hit a pin or 2 whilst bowling at MFA Bowl and became Quiz Champions of FMS!

Another highlight was the PAN!C Bake Off, where I almost fell into a sugar induced coma!….. mmmmm cake. And along the way we raised a massive £361.56 for charity (which was split between Macmillan, Doctors without Borders and Age UK).

Fabulous cakes at the PAN!C Bake Off, September 2014

Since forming, the PAN!C committee has had 10 members, and I’d personally like to thank every one of them for contributing and making this association such a fantastic and fun thing to be a part of. I’d also like to thank the ICaMB postgraduate students too. We’re a student led committee for the students and without you we’re nothing!

I’m nearing the end of my stint at Newcastle University, so it’s time to pass the PAN!C reigns over. It’s something that has improved my time management and helped me to focus on my PhD work. I hope you too realise the opportunity that PAN!C offers. You can give as little or as much time as you like, involve yourself with conference organising or just attend the socials. So, get involved and let’s keep the party going!

If you want any further information or just to chat about PAN!C, please don’t hesitate to contact me: b.m.lawry@ncl.ac.uk.

ICaMB – Inside Cells and Molecules Blog

Monthly Archives: March 2015

When a Teardrop Can Kill: The Deadly Ebola Virus

What did the Black Death ever do for us? The curious route to an Industrial Biotechnology Catalyst Grant

Happy Birthday PAN!C