Fighting cancer with a virus of plants: it's not a load of bull

If I ask you to think of a virus, I’m going to guess you’ll land on one of Zika, Ebola, HIV, Influenza or maybe at a push, Rhinovirus, the latter two of which you may be unfortunate enough to have recently experienced in the winter of the northern hemisphere. Viruses, and the infections they cause, are massive public health concerns, and for the most part this is what gets our attention. However, there is currently a vast amount of research going into the development of viruses as tools in the fight against cancer, and it’s some of this work I’d like to discuss in the blog post.

The work I’m going to talk about was published in the journal Nature Nanotechnology and comes from the Geisel School of Medicine in New Hampshire, USA. The published article has the somewhat striking title of “in situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer.”

Let’s break that down. Our immune system has the capacity to destroy cancer cells, this is in fact one of the major hurdles a mutant cell needs to overcome in order to develop into a cancer. In order to escape the immune response, mutated cells need to develop ways in which to protect themselves, for instance, by releasing molecules that act to suppress the the immune system - in a sense, camouflaging themselves. This kind of immunosuppression by cells is nothing strange in our body, it’s part of a normal response to stop your immune system attacking your own body. However, cancer cells develop the ability, through mutation, to subvert this system to aid their own growth. As such, an idea that has starting to come to the front of cancer research is so called cancer immunotherapy; stimulating the immune response to fight against cancerous tissue, while leaving normal tissue untouched. So to break down the first part of the paper’s title, “in situ vaccination” is the idea of directly supplying something to trigger an immune response at the site of a cancer. The “something” that is being supplied into cancerous tissue in the case of this work, is cowpea mosaic virus nanoparticles.  

 

Cowpea peas

 

I’m guessing that cowpea mosaic virus isn’t something you’ve come across before (I certainly hadn’t). By way of background, cowpea mosaic virus, is a virus that infects the cowpea plant, and causes a mosaic pattern on the leaves (to state something you’d probably guessed). In this study of cowpea mosaic virus as an anti-cancer agent, the group work with “nanoparticles," which can also be called "virus-like particles." If you consider a virus particle, such as the one depicted for cowpea mosaic virus (below), that 3D shape is made up by very few proteins. A virus structure is essentially just a protein case for the genetic material. A virus-like particle is simply just this casing with nothing inside (literally a shell of it’s former self). Without the genetic material, these particles are no longer able to infect cells. An analogy for this could be a laptop that has had all of the internal circuit boards removed. From the outside it still looks like a laptop, but try and turn it on, and nothing will happen. Virus-like particles are of very little danger because they cannot infect cells, however, the immune system isn’t able to detect this. The cells of the immune system only ever see the outer casing of a virus and treat this as a threat - thus virus-like particles can stimulate an immune response just like a real virus (from the outside a laptop without circuit boards still looks like a laptop). And this is the basis of some vaccines, most notably of which is the vaccine against human papillomaviruses, which some readers may have received. 

 

Cowpea mosaic virus structure (from Wikipedia)

 

We can now get to the meat of the paper. To jump to the punchline, the group were able to show that injecting mice with cowpea mosaic virus-like particles could stimulate an immune response, that resulted in suppression of cancers in mice. Initially the group determined that treating cells with the virus-like particles was capable of triggering an immune response in a dish, and moreover, found that this treatment didn’t cause the cells to die. The group moved on to have mice inhale preparations of cowpea mosaic virus-like particles. In mice with no tumours, this inhalation resulted in activation of the immune system, which could be detected by an increase in the number of neutrophils (one class of immune cells) in the lungs. And indeed, collecting neutrophils from these mice showed that the virus-like particles had been taken up into these cells, thus activating them - the neutrophils treated the virus-like particles as a true threat and attempted to remove them. Importantly, this inhalation of the virus-like particles to healthy mice didn’t cause any adverse side effects or damage to the lungs.

The next experiment was to give the virus preparation to mice with tumours. It was found that before the inhalation, the immune cells in the lungs of these mice was very different to that of healthy mice. There were far more cells associated with immunosuppression (as a result of the cancer). Upon giving virus-like particles by inhalation to these mice, it was seen that there was a large increase in the number of activated immune cells capable of tackling cancer (again, a large increase in the number of neutrophils, for instance). So far then, everything seems to be going to plan for using cowpea mosaic virus to activate an anti-cancer immune response.

Next, the group started to give mice injections of the virus-like particles instead of allowing them to inhale. They found that injecting mice with cowpea mosaic virus-like particles resulted in a decreased number of tumours in the lungs. Importantly, the researchers nicely demonstrated that this reduction in tumour number was due to an immune response but using mutant mice that don’t have a functional immune system - these mice showed no change in their tumour burden.

The tumours that were being tested to this point were melanomas growing in the lungs. But it wasn’t just melanomas that showed sensitivity to the immune response triggered by cowpea mosaic virus-like particles. In one set of experiments, mice were injected with cancerous tissue in their fat pads, which then spontaneously metastasis to the lungs 16 days later. The mice given injection of virus-like particles all showed delayed onset of lung tumour formation, and survived the treatement. Similarly, mice with tumours in their colons could also be protected by treatment with the virus-like particles. 

Finally, the group tested whether injections of virus-like particles could protect against melanomas growing in the skin. Once again, they found that cowpea mosaic virus-like particles could protect the mice, and half of the mice injected completely cleared their tumours after only two injections of virus preparations. Moreover, this anti-tumour immune response was found to be long lasting, since when mice that had previously cleared tumours were injected with more cancerous tissue at a later date, 3 out of 4 completely rejected the implanted cancer (while this cancer grew in all non-vaccinated mice). 

Overall this work has nicely demonstrated the potential for the use of cowpea mosaic virus-like particles in the battle against cancer. These virus-like particles could be tolerated in mice and were seen to trigger an immune response, which resulted in clearance of cancerous tissue. The virus-like particles showed no major side effects, and even seemed to provide long lasting immunity against the cancers used in the work. The next step for cowpea mosaic virus will probably be to test safety in humans. It will take a long time, and many more studies, but perhaps a virus that infects plants could one day be used to treat cancers in humans.

From nowhere to everywhere - Zika virus

It seems to have come out of nowhere, and spread like wildfire. A new virus is rampaging through Brazil and parts of the Americas, and causing major global concern. Just as Ebola finally subsided in Africa, enter Zika virus from left stage. I’ll be honest, until a couple of weeks ago, I’d never heard of Zika virus, and I’m probably not alone. So I’ve decided to put together this blog post to discuss some aspects of this virus and the current outbreak. 

 

Countries where Zika virus has been found - from the New York Times

 

To being, let’s look at the virus itself: Zika virus is a member of the Flaviviridae family, which includes the much better known dengue, West Nile and yellow fever viruses, and like these is transmitted by mosquitoes. Because no-one really cared about Zika until recently, the virus has little published scientific literature. Typing "Zika virus" into PubMed (the major search engine of academic literature) yields 155 published articles, compared to the 9198 that are returned from searching "dengue virus” (at the time of posting). However, being that we know the virus family, this already tells us a lot. Like other flaviviruses, Zika is small with a genome of just 10.8 kilobases of positive sense single stranded RNA, which can encode 10 genes. The genome being positive sense and single stranded means that once it is released into a cell, the RNA will be treated just like any other cellular, protein encoding, RNA and interact with ribosomes to produce new protein. The 10 proteins that are produced by a cell following a Zika virus infection are responsible for taking over that cell and replicating the virus so it can spread new particles to new cells. Again, as with other flaviviruses, the Zika particle is small, and has one protein, the E protein, protruding on the surface. This protein is involved with attaching to a cell and responsible for the subsequent events that culminate in release of the genome into that cell. The E protein also makes up the main target for the immune system to tackle the virus. 

 

The structure of a flavivirus - from ViralZone

 

Zika virus was first identified in 1947 from a Rhesus macaque monkey which was being used as sentinel for yellow fever. At the time, sentinel monkeys were commonly used to detect the presence of yellow fever in an area since tests for viruses weren’t quite what they are today (at the time there weren’t even cell line to use in a lab to infect with the virus to study it!). This particular monkey, Rhesus 766, developed signs of a viral infection while in the Zika forest of Uganda. Blood was taken and used to inoculate mice, all of which became sick. Virus was isolated from the brains of deceased mice, but determined to be different from yellow fever - thus Zika virus was defined. The first human cases of the viral infection were reported in 1968 from Nigeria. Zika virus then remained largely undetected outside of Africa and parts of Asia, until 2007 and 2013 which saw epidemics in Yap Island and French Polynesia, respectively. 

Zika virus generally causes very mild symptoms in human infection, if any symptoms at all - only about 25% of cases are symptomatic. For that reason, little attention had been paid to it, until recently of course. The 2013 outbreak in French Polynesia was the first real indication that Zika infection could have more serious consequences than just rash, fever, malaise, and all the other general symptoms the initial infection can cause. During the outbreak, a spike in the cases of Guillain–Barré syndrome (GBS) were reported, with 73 individuals being diagnosed with the disease. GBS results from damage to the perisperhal nervous system (i.e. not the brain or spinal cord), causing rapid onset muscle weakness, and in severe cases, can result in paralysis. GBS can be life threatening since the peripheral nervous system is responsible for controlling the muscles involved with breathing and the heart beat. This coincidence of Zika virus spread and GBS was the first indication that Zika could potentially damage the nervous system.

So that brings us to the current outbreak occurring in Brazil, where once again, there appears to be circumstantial evidence linking Zika virus to damage of the nervous system - this time in the form of microcephaly in new born infants. Microcephaly, meaning abnormal smallness of the head, can be caused by multiple factors. Other infections can be responsible, such as rubella virus, cytomegalovirus or toxoplasmosis. As can poisoning of a foetus from alcohol, mercury or radiation. Mother malnourishment and diabetes may also be linked to microcephaly. In about 15% of microcephaly cases, the infant just has a head of smaller size, but in other cases, this small head is connected with poor development of the brain which can result in developmental delays, intelligence deficits and hearing loss, as well as premature death.  

 

Microcephaly - from Wikipedia

 

In the previous paragraph you may have noticed my wording that there is circumstantial evidence linking Zika virus to microcephaly cases; it is not clear that Zika infection is responsible. However, as Zika has begun to spread in Brazil and parts of the Americas, there has been a phenomenal spike in the number of microcephaly cases. Until 2014, there were around 150-200 cases of microcephaly in Brazil each year. The birth index for Brazil is estimated at 14.72/1000 population, in a country of around 200 million, this means just under 3 million as an estimate for the number of births in Brazil each year - so cases of microcephaly were rare. However, in 2015, there were nearly 3000 microcephaly cases, with the vast majority of these being reported in the latter part of the year, right around the time Zika virus cases started to become more common.

As I’ve stated, it is still not clear that Zika virus is responsible for the rise in microcephaly cases, though it is clearly the major culprit. More time is needed to follow pregnant women and determine if they become infected with the virus during their pregnancy. However, there will still be difficulties to determine if Zika alone is responsible. The best way to confirm a Zika virus infection is to look for the viral RNA genome in the blood of patients, however, the time in which this can be done is limited to the first week of infection. After that, diagnosis relies on finding antibodies against the virus in a patient’s blood. However, these serological tests are complicated by the fact that antibodies against Zika virus can cross-react with dengue virus - making it difficult to tell which virus was truly responsible for the presence of those antibodies. That dengue virus is endemic in Brazil, and the vast majority of people are positive for antibodies against dengue, compounding the issues with truly detecting Zika virus. However, with studies now aimed at determining if Zika virus is truly responsible for microcephaly, pregnant women will be tracked much more carefully, therefore raising the prospects of being able to catch an infection in the first week where viral RNA can be used to confirm a Zika virus infection.

Finally, what can be done? The short answer is not a great deal. We currently have no antiviral drugs to tackle any flavivirus infection, let alone Zika. Work is under way around the world trying to find broad-spectrum antiviral drugs that could be used to combat infection from multiple viruses, but whether a drug intervention would be of much use in a disease that generally causes few symptoms, except in pregnant women who may be unable to take the drugs, remains unclear. A better prospect would be the development of a vaccine, but we are a long way from that. Vaccines to Ebola were starting to undergo trials towards the end of the recent West Africa outbreak, but these had been years in the making through research on Ebola. Very little study has gone into Zika virus and potential for vaccines against it. However, there is a vaccine against yellow fever virus, and one idea would be to modify this to have the E protein of Zika, instead of yellow fever. The yellow fever vaccine is one of the best vaccines we have developed, but generating a Zika vaccine, and testing it, will take many years. Making things worse is the ethical issues of testing the vaccine in the population that seem most at risk from Zika virus, pregnant women. 

 

 

For the time being, our best option may be to tackle the mosquitoes which transmit the virus. There are the basic measures of wearing clothes to cover the skin and the use of bed nets when sleeping. Additionally, clearing standing water pools, such as in flower pots, or the inside of old tires, where mosquitoes lay their eggs will help to disrupt the mosquito life cycle. And in terms of more complex measures, many tests are under way to release genetically altered male mosquitos which cannot produce viable offspring. Additionally, work on the bacteria Wolbaccia may still hold hope for reducing the risk posed by mosquitoes (this is something I’ve previously posted on). Tackling mosquitoes seems to be one of the biggest challenges we face in the coming years. The Aedes species, responsible for transmitting Zika virus also transmit dengue virus and Chikungunya virus. The Culex species transmit West Nile virus, and the Anopheles species transmit malaria. Along with global warming, the habitable zones of these mosquitoes will spread, putting more people at risk. The best way we can tackle the dangers posed by mosquitoes is through continued scientific research, both on the mosquitoes themselves, and the viruses they harbour.

The most pressing issue now for Zika virus is determining that it is indeed the virus that is causing microcephaly, and finding ways to tackle its spread by mosquitoes.