How do viruses hijack our brains to make us vomit - and can we stop it?

Human rotavirus particles - http://faculty.riohondo.edu

We've all experienced it; that is, the awful unwell sickness that overwhelms you when you've picked up a nasty viral infection. Remember the last time you had a cold or the 'flu -  when coughs, headaches, sore muscles, vomiting, diarrhea and general fatigue forced you to remain in bed. Yet, just exactly how does the virus manage to do this to you? Especially when in some cases your sickness allowed the virus to spread from you to an uninfected family member; sometimes there is an evolutionary advantage to making you unwell. A recent paper published in PLoS Pathogens this week (which can be read here) investigates the molecular mechanisms behind why the oft' fatal rotavirus causes those infected to vomit and through doing so sheds light on possible therapeutic strategies. 

A gut villus . http://rezidentiat.3x.ro

This research isn't just aimed at answering some obscure academic problem - although it does do it - rotavirus induced gastroenteritis is serious business worldwide, with over half a million deaths each year (and many, many more hospitalised) mostly due to severe dehydration caused by the excessive vomiting and diarrhea. In many countries, the rotavirus vaccines just haven't reached the population at large and the reasons why this virus causes vomiting and diarrhea are poorly understood - hence this paper.

Now, vomiting - like many physiological processes - is controlled by our brain. Usually vomiting is induced by eating something nasty and is hence a kind of defense mechanism to rid ourselves of any harmful things within the gut. In order to sense if there is anything bad in your intestines, the brain is wired up to it's surface through what is known as the enteric nervous system or ENS. This mesh of neurons is connected to particular endocrine sensor cells (see left), called enterochromaffin cells (EC cells) that line our intestines and respond to the harmful stuff through the release of chemical messengers - like serotonin (5-HT), which in turn activate the ENS and end up inducing vomiting and diarrhea via our brain. But, specifically how does rotavirus fit in here?

 A straightforward hypothesis here would be that following ingestion of rotavirus, it would infect the EC cells lining the gut and somehow alter them to release serotonin and stimulate vomiting/diarrhea. And so, the used a primary EC tumour cell line to show that rotavirus does indeed infect the cells. So, now that the virus infects the cell, is this how it causes induction of the ENS? A rapid induction of calcium release was observed soon after infection as well as a quick secretion of serotonin - which itself is dependent on calcium signalling yet the fact that this release occured so soon after addition of  virus suggested that replication was not responsible for it - maybe something in the virus particle or something that was in the solution with the virus was to blame? Following seperation of virus particles from the culture media used to grow it, the group showed that the virus particles themselves were not behind it. The group tracked this effect down to NSP4, a protein secreted from rotavirus infected cells following infection and would therefore have been carried over with the virus in these experiments. In the end it was this protein itself that was responsible for the induction of calcium signalling and downstream serotonin release. 

Gut villi from mice infected with rotavirus. Arrows indicate rotavirus infected cells (those with large vacuoles) and stars indicated EC cells stained brown. Below, red = rotavirus and green = serotonin and yellow = colocalisation or a rotavirus infected EC cell. Yellow cells are rare - non-EC infected cells (enterocytes) are not.

All done and dusted now? No - up to this point, all work was done under tissue culture conditions in vitro - all be it, mostly in primary cells. The group then moved on to a small-animal mouse model of rotavirus infection to determine if this was the case in vivo - does rotavirus infect EC cells and induce calcium/serotonin signalling that stimulates gut neurons to induce vomiting/diarrhea. This could not have been addressed in cell culture. Following mouse infection, cross-sections of the gut wall were studied to determine the location of rotavirus infected cells and EC cells (see above). Many of the enterocyte cells lining the mouse gut were infected while only rarely were the EC cells targeted - despite what was observed in vitro. Yet for this model to work, rotavirus did not require infection of EC cells as NSP4, the protein behind this effect is secreted from cells and could hence stimulate EC from a distance. In the mouse, the group show that serotonin administration results in diarrhea (oddly, mice do not - and cannot - vomit) and also rotavirus infection leads to the activation of regions in the brain associated with sickness in humans (done through fos staining of brain sections).

A model for how rotavirus makes us vomit/diarrhea

This work has built up a kind of nice and simple picture of a mechanism of rotavirus causes vomiting and diarrhea. They have shown that the virus is able to enter and replicate inside epithelial cells and endcocrine cells lining the gut; they showed that this led to the release of serotonin - the stimulator of vomiting/diarrhea - and that this was down to NSP4 synthesis and release and finally that mice infected with rotavirus activated brain regions associated with vomiting and diarrhea.

The model: (see above) - Rotavirus infects gut cells - expresses and releases NSP4 - NSP4 stimulates calcium signalling and serotonin release from nearby EC cells - serotonin stimulates close by neurons that activate vomiting/diarrhea areas of the brain, which causes serious fluid loss, dehydration and subsequent death. Also this results in rapid spread of virus particles and transmission of infection.

The good thing is now, with this mechanism at hand, we may be able to partially inhibit this response and save the lives of many children and prevent the spread of rotavirus in these populations. Luckily for us, a number of generic - and hence cheap - already in use anti-sickness tablets (serotonin receptor antagonists) exist and could be applied in this setting. Although previous research had established the efficacy of using this drug to attenuate diarrhea in rotavirus infections, this paper established a working mechanism for its activity.

Granisetron - a serotonin receptor antogonist used as an anti-sickness medication and could be developed to aid rotavirus-induced vomiting/diarrhea. http://upload.wikimedia.org/wikipedia/

Hagbom, M., Istrate, C., Engblom, D., Karlsson, T., Rodriguez-Diaz, J., Buesa, J., Taylor, J., Loitto, V., Magnusson, K., Ahlman, H., Lundgren, O., & Svensson, L. (2011). Rotavirus Stimulates Release of Serotonin (5-HT) from Human Enterochromaffin Cells and Activates Brain Structures Involved in Nausea and Vomiting PLoS Pathogens, 7 (7) DOI: 10.1371/journal.ppat.1002115

Vaginal 'probiotics' may protect against HIV transmission

According to WHO data, 2009 saw 33.9 million people worldwide infected with the human immunodeficiency virus -  HIV and of these, 1.8 million died from AIDS while 2.9 million were newly infected with the virus. This leaves a year-on-year increase of just over 1 million HIV positive people of which, many of these will go on to pass the virus. Therefore any strategy to eliminate HIV from the human population will have to aim at both treating those already infected as well as preventing new viral transmission and if this is achieved, HIV infection worldwide would dramatically decrease with every year, severely reducing the global health burden caused by this viral pandemic. The only problem is, how can we successfully prevent transmission?

Despite a significant decrease the number of people infected by HIV continues to grow year on year

HIV entry and pathogenesis - where and how can we target it?

One of the major routes of HIV transmission is through sexual activity: an HIV positive person will be carrying many copies of the HIV genome within their cells and in turn these will be able to generate new infectious virus particles. These virions - either present in blood or semen - may be mechanically transferred from infected to uninfected people during sexual activity and many immune cells (Langerhans cells, macrophages and intraepithelial CD4+ T cells to be precise - see below) lining and within the mucosal epithelial surfaces of the reproductive tract are the initial target cells for HIV entry into the human body. It is here that the cells are exposed to the virus through sexual contact, facilitating virus uptake and initial infection then allowing the virus to spread within the body and potentially set up the chronic infection that may develop into AIDS.

HIV entry via immune cells within the vaginal mucosa and spread to systemic lymphoid organs. Miller, 2007.

Many potential strategies are currently in development which aim at preventing person-person transmission during sexual activity through the inhibition of HIV particle transfer. This is the reason why condoms - male and female - are so effective. As the authors of the paper outlined below highlight the requirements of such a strategy:

In many cultural settings, women need a product that can be used covertly without obtaining the permission of their sexual partner. In addition, the cost of HIV prevention must be affordable to the developing world. Thus, there is still a need for products that block HIV transmission, are safe and easy to use, and are coitally independent, discreet, and cost effective.

Bacterial symbiosis to the help

All being so, our bodies are not completely defenseless when it comes to preventing sexually transmitted infections, including HIV; we have multiple tricks up our sleeves and one of which is through a form of bacterial symbiosis. The human reproductive tract is covered in a bacterial biofilm composed of a few species of bacteria, predominantly Lactobacilli. These microbes regulate vaginal biology and aid in the protection against variable infections through the formation of a physical barrier and through alterations in pH. But of course it isn't enough and as I mentioned earlier, people are all too often getting infected with HIV. Yet what if we could enhance these bacterial defenses through genetically engineering those bacteria that natural colonise the reproductive tract? This is exactly what a recently published paper in Mucosal Immunology reports -  the development of a novel genetically engineered live bacterial strain expressing an anti-HIV protein that can be easily applied to the vagina and prevents HIV transmission

Normal tissue properties A) and Presence of anti-HIV protein CV-N

The group had previously generated an engineered strain of Lactobacillus jensenii (termed:1153-1666) that expressed and secreted a modified Cyanovirin-N (CV-N) protein. This protein has been shown to have a broad inhibitory activity against a range of HIV-1 strains and provide protection from infection in a non-human primate model of HIV through inhibiting virus entry into those target immune cells - it is also highly potent and non-toxic. To establish the potential for administering this bacteria in humans, the group inoculated the vaginas of non-human primate macaques on a regular basis and achieved stable colonisation along with expression of the antiviral protein in the fluid lining the tract; the recombinant L. jensenii was detected along the epithelium while no tissue changes were observed and no untoward inflammation was detected, all indicating the safety of this strategy.

in vitro HIV inhibition with non-recombinant (left) and recombinant (right) bacteria

So far, the ability of this recombinant bacteria to inhibit HIV infection has not been addressed so later it was assessed through the use of an in vitro model that accurately portrays the physical and biological architecture of the human vagina as well as the macaque model that uses chimeric human/simian immunodeficiency virus infection. Colonization of the in vitro vaginal tract with the non-recombinant bacteria resulted in a 23% reduction in HIV infection compared to the control while inoculation with the CV-N expressing strain gave 72% inhibition (see above). Groups of macaques were then treated with an antibiotic to remove any endogenous Lactobacillus colonizing their vaginal tract and were subsequently inoculated repeatedly with the recombinant strain. These monkeys were then repeatedly challenged with the virus in a manner similiar to what would happen under natural human conditions and the ability of the virus to infect each animal was assessed over time. This strategy reduced the infection rate by nearly 63% (see below).

Macaque challenge outline and results of bacterial colonization protection

 This group has generated a bacterial strain - closely related to that already present within the human reproductive tract - that has been genetically augmented through the introduction of the gene for a potent HIV-inhibitory protein Cyanovirin-N. This recombinant bacteria can be inoculated into the vagina and affords protection against initial HIV infection in an in vitro and in vivo macaque model through the expression and secretion of CV-N without generating a toxic response; this engineered microbe is thus better at preventing infection that the non-CV-N expressing bacteria. It remains stable over time But, how then would this function in the real world?

  In order to halt the HIV pandemic, we require an at least partially effective strategy to prevent person-person transmission of the virus and this method must safe and inexpensive if it is to be administered to the many people who require it across the developing world. This method outlined above - pending the results of further clinical work - highlights the importance of novel strategies to combat HIV spread. The group outlines the development of an easy-to-apply, safe, efficacious and cheap method to combat HIV transmission in two model systems. Despite these pleasing results, further work will need to be carried out in a clinical field trial to determine whether this mode of protection will work in the real world.


Lagenaur, L., Sanders-Beer, B., Brichacek, B., Pal, R., Liu, X., Liu, Y., Yu, R., Venzon, D., Lee, P., & Hamer, D. (2011). Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus Mucosal Immunology DOI: 10.1038/mi.2011.30

Part II - why vaccine viruses are weaker that their pathogenic cousins

I've talked about this before (here with mumps virus) but just what makes vaccines so good at being vaccines? - that is, what makes them safe yet immunogenic? The great thing about this is that maybe if we understand these processes a bit better we may even be able to develop safer, more effective and cheaper vaccines that will ultimately save more and more peoples lives. A lot of research at the minute is cuurently attempting to tease apart the roles of hundreds of virus genes in infection and disease with many groups focusing on specifically how these genes affect attenuation, that is how they make the virus weaker and less dangerous so it can be used as a vaccine.

So it starts off like this: we know vaccines are safe and effective when after we give them to people/other animals very little get sick and a lot of them are immunized against infection with that particular virus yet the 'wild-type', normal virus is able to infect and cause disease - so just what makes these different? Well to find out we have to compare the two viruses - the weak one and the virulent one and somewhere in their genomes lie differences that will possibly shed light on the important molecular mechanisms behind virus attenuation and pathogenesis.

Canine distemper in dogs. http://www.dogs-info.net

In a recent paper, Dietzel et al investigated these processes using a great model system, specifically canine distemper virus - or CDV, a virus able to infect and cause life-threatening disease in a whole range of mammalian species in a domestic or wild-life situation and hence is obligatorily given as part of a multivalent vaccine to all domestic dogs. The group began by comparing the matrix - or M- gene between a vaccine strain of a virus known as canine distemper virus or CDV and a wild-type one. This M gene encodes a protein which plays a major role in the assembly of new virus particles, cell-cell spread and particle stability within CDV-infected cells through co-ordinating genome interactions with virion-surface proteins. Its role in CDV pathogenesis - and other viruses - has barely been looked at. The two M proteins differed at only six different amino acid positions (3%) so to look into the role of them both during CDV infection the group genetically engineered a CDV virus based on the wild-type but carrying the vaccine-derived M gene in place of its own. The growth and physical characteristics of the three viruses :wild-type, wild-type + vaccine M and vaccine strain viruses were looked at as well as physically characteristics of the produced particles, how they are assembled and what happens during animal infection.


The 3 viruses grow equally well in cell culture conditions.


Despite the three viruses growing equally well under cell culture conditions (see growth above and how the infection looks in tissue culture - all are pretty much similiar), the two differed markedly in their particle-infectivity ratio, that is - the number of virus particles capable to carry out a successful infection compared to the total number of particles in a given volume, i.e some particles may be non-functional for some reason, maybe because they are less physically stable or damaged.

virus particle release from the tops or bottoms of the cells

The vaccine virus also differed in the way new CDV particles were released from cells: the wild-type virus is released from the tops of polarised epithelial cells while the vaccine strain is released from both the top and the bottom. However, transfer of the vaccine M gene wasn't able to confer bipolar release onto the wild-type virus indicating the function of other CDV genes in this phenotype. To determine a mechanism for this bipolar release the group analysed the intracellular distribution of the key CDV proteins, H and F involved in particle production and cell-cell spread. They found that this proteins differed in their location between the three viruses: the wild-type only had them on the tops of cells while the vaccine strain had them on both sides, the chimeric virus had an intermediate distribution. These results highlight the primary ability of the CDV matrix protein to coordinate protein distribution within the cell.

CDV protein intracellular distibution

All these results are good and all but what effect do they actually have in vivo - in a situation where the virus is infecting multiple cell types and is constantly battling the immune system? To determine the effect of vaccine M gene, groups of ferrets (an excellent model of CDV infection that are naturally infected by the virus) were inoculated intranasally with the three viruses and disease severity, body temperature, disease symptoms and fatality was recorded. Interestingly, based on these measures, the wild-type with the vaccine M was more attenuated that the vaccine virus as shown below with the measure of ferret blood leukocytes - the more blood cells the less pathogenic the virus.

A measure of CDV disease - the percent of ferret blood cells - a CDV target cell


The process of pathogen attenuation for the generation of protective vaccines has worked well in the past for many diseases yet many viruses and bacteria do not prove to be responsive to this method. Other more targeted processes may have to be designed in order to create vaccines for against these diseases. Work investigating the molecular basis of virus pathogenesis and attenuation may allow us to rapidly and successfully attenuate these hard-to-vaccinate viruses in a more targeted fashion - and one that may work better than the previously tried methods.

Dietzel, E., Anderson, D., Castan, A., von Messling, V., & Maisner, A. (2011). Canine Distemper Virus Matrix Protein Influences Particle Infectivity, Particle C
omposition, and Envelope Distribution in Polarized Epithelial Cells and Modulates Virulence Journal of Virology, 85 (14), 7162-7168 DOI: 10.1128/JVI.00051-11


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How come vaccine viruses are so safe while normal viruses are so dangerous?

One method that has been used extensively to generate worthwhile vaccines is that of forcing an initially disease-causing virus to replicate inside a non-natural cell, for example imagine forcing the human specific measles virus to replicate within cells from a chicken. Over time these viruses - all with extremely high mutation rates - will evolve and adapt to the conditions within a chicken cell while at the same time losing it's ability to survive within human cells. The use of these live-attenuated viruses as vaccines has led to the dramatic reduction in a number of important human - and livestock - viruses - the likes of measles, mumps, polio and rinderpest. 

These vaccine viruses usually retain their ability to infect and replicate within their host (one of the reasons why they are so good at protecting us) yet fail to cause significant disease past the odd fever. However, in some cases the use of these vaccines has led to a number of cases where they caused serious illness. Hence, with the powerful immunity these vaccines generate comes the important potential chance of causing disease and therefore the ability to understand and predict how a particular vaccine will behave following administration is key to continuing the safe use of live attenuated vaccines. Sadly, our knowledge of the mechanisms behind virulence and attenuation are largely unknown.

Vaccination of the man behind the mumps vaccine - Maurice Hilleman's daughter, Kirsten, while her half-sister, Jeryl Lynn, looks on. The MuV vaccine strain Jeryl-Lynn was originally developed from the virus which infected Hilleman's daughter of the same name through non-natural replication. http://www.historyofvaccines.org/

One example of this can be found with the case of the mumps virus (MuV) vaccine. Interested in mumps? see here. Before the widespread use of the vaccine, MuV was responsible for the majority of cases of aseptic meningitis (inflammation of the lining of the brain not caused by bacteria) in the western world - this virus is highly adapted in entering the human central nervous system and replicating within the epithelial cells that line its inner layer, making it particularly dangerous. Because of this, any vaccine batch produced must go through rigorous pre-clinical testing, the majority of which is carried out in primates. Yet sometimes, vaccine viruses will lead to the development of aseptic meningitis following immunization. We still do not adequately understand the molecular basis of the ability of MuV to cause disease in the nervous system - something that may facilitate the development of new and improved MuV vaccines.

Sauder et al, publishing in Journal of Virology, explore the genetic basis of MuV neuropathogenesis through the generation of around 30 chimeric viruses - see below - comprising genes of a neurovirulent MuV (strain: 88-1961) and a highly attenuated MuV (vaccine strain Jeryl-Lynn 5). Through the analysis of the potential for these viruses to cause disease in a rat model of mumps meningitis they were able to assess the contribution of specific genes - or combinations of genes - in either increasing or decreasing its ability to cause disease.

Chimeric mumps viruses (combinations of attenuated - JL and virulent - 88). What effect will each have on pathogenesis?

MuV has a single-stranded RNA genome of 15,384 nucleotides and encoded within this one molecule are 7 genes through which at least 9 different proteins are expressed.  These proteins - and hence their corresponding genes - govern the basic biology of this virus: building of the virus particle, receptor binding, cell entry, transcription and replication and finally, cell exit. N, P and L forming the replication apparatus of the virus while M, F, SH and HN are involved in particle assembly, entry and exit. It is these same proteins that are responsible for the ability of MuV to cause disease in humans, specifically aseptic meningitis. Any understanding of the ability of a virus to cause disease must identify its key molecular - and genetic - components hence, what particular genes along the MuV genome are responsible for causing aseptic meningitis in humans? Is it those that allow the virus to enter the cell? Those involved in replication? or those involved in building the virus particle? Sauder et al sought to try and convert an attenuated virus into a virulent one and vice versa - and in doing so uncover the biology behind MuV pathogenesis.

Can we transform the virulent 88 strain to an attenuated virus by inserting combinations of attenuated JL genes?

Above shows the results from the initial experiment comparing disease caused by the different viruses: This is where the added different genes from the attenuated virus to the virulent to see whether or not this weakened the viruses ability to cause disease. As you can see, no individual genes or combinations added to the 88-1961 virus caused it to be as attenuated as the vaccine strain, suggesting that in this case with MuV attenuation is a complex, polygenic trait involving many genes. Neither the transfer of all replication proteins (N, P and L) nor the assembly proteins (M, F, SH and HN) resulted in complete attenuation. The most dramatic effect was seen with N and M transfers - although the reason why was not addressed. 

Can we transform the attenuated JL strain to a virulent virus by inserting combinations of pathogenic 88 genes.

Following on from this, the group tried to see whether they could turn the attenuated into the virulent virus through carrying out the reverse of the above experiment although this time the results were not the same as no genes or combinations resulted in anywhere near the levels of pathogenesis seen for the 88-1961 virus. Even with the addition of the previously effective N and M combination.
 

These results tell us a number of things about MuV attenuation and virulence, firstly: it's a lot more complex than we might have previously thought! - this is not all down to one gene but a few of them working together in combination. Secondly, it doesn't work both ways - the mechanisms behind how a virus causes and doesn't cause a disease are different as the same genes couldnt do both. And lastly these results point us to some interesting avenues of future research into virus attenuation - what are the specific molecular roles each of these genes play in turning the virulent virus into an attenuated one? For example, why do the attenuated N and M have such a drastic effect? More work will be undoubtedly be done to determine these questions and this may allow us to rationally attenuate viruses in the future using these combinations of genes.

Sauder CJ, Zhang CX, Ngo L, Werner K, Lemon K, Duprex WP, Malik T, Carbone K, & Rubin SA (2011). Gene-specific contributions to mumps virus neurovirulence and neuroattenuation. Journal of virology, 85 (14), 7059-69 PMID: 21543475

Can we use viruses to vaccinate against - and cure - established cancers?

Everyone is aware of the ability of our immune system to defend against microbial pathogens yet its role in the prevention of other diseases - like cancer - is generally over-looked. Yet it is through the harnessing of our immune system that novel ways of combating cancer may arise. And interestingly enough, through the use of engineered viruses - the same ones our immune system protects against - we may now control aspects of immunity to suit these medical needs. Kotke et al, report in Nature Medicine just this week, their use of a new virus-based immunotherapy platform that was able to effectively 'cure' mice suffering with cancer.

One of the hallmarks of cancer appears to be the ability to persist in the face of an active immune system - see fig. 1. Newly cancerous cells and tumours are able to survive and proliferate without - or at least protecting themselves - against a full-blown immune attack. Our immune system is usually able to protect us from the development of cancer but in some cases something fails and the result is more often than not - cancer.

Fig 1. Newly recognised cancer hallmarks - note avoiding immune destruction. (Hanahan and Weinberg 2011).

Through the recognition of 'tumour antigens' (proteins expressed only on cancer cells) or 'tumour-associated antigens' (proteins expressed differently on cancer cells), our immune system is usually able to mount an effective response toward those cells. This is the  system that tumours are able to suppress yet we may be able to boost the natural immunological nature of tumours in order to cure them. The discovery of these proteins - just like those found on the surface of virus particles or bacterial cells - may allow us to effectively vaccinate people against cancer, allowing their own immune system to remove the cancerous cells.

This is what we try and achieve through cancer vaccines and immunotherapies. Kotke et al set about trying to improve upon these current immunotherapy platforms, which - as they state - suffered from a number of problems including: lack of known tumour antigens and coverage of only a few such proteins. Most current immunotherapies rely upon the immunization with only a single antigen. Previous work by the group showed that if you kill normal cells from a patient in vivo - you may be able to elicit an effective anti-tumour immune response through the induction of tumour-associated antigen immunity (when cells die they either burst and release their insides).This work effectively showed that you could immunize with a wide range of tumour-associated antigens from normal cells and protect against cancer - both circumventing the above two problems.

VSV particles - www.standford.edu

To improve upon this model, they developed a virus-based platform for the expression of a wide range of tumour-associated antigens in vivo termed altered self antigen and epitope library (ASEL) - see fig 3. They based their method upon the vesicular stromatitis virus - or VSV - normally a virus solely of livestock that also has the ability to infect humans. In humans it causes a generally mild flu-like illness and may form vesicles on the skin. Although a single-stranded non-segmented negative sense RNA virus, we have the ability to generate infectious VSV particles entirely from cDNA plasmids encoding the entire VSV genome. This has facilitated the development of VSV as a key eukaryotic expression vector for multiple uses, such as these cancer immunotherapies and specifically, as an oncolytic treatment. Using standard molecular biology techniques (PCR, restriction enzyme digests and ligations) you can insert any gene from whatever source you want into the VSV genome and it will be expressed inside cells following infection. The benefit with using this virus is that even without the expression of tumour antigens from it's genome, replication within a cell will kill the cell anyway. It is a double hit strategy.

Fig.3. Cloning the cDNA library into the VSV genome in forward and reverse orientations = VSV-ASEL library

In order to express hundreds of tumour-associated antigen genes, the group used reverse-transcriptase PCR to amplify all expressed genes from normal prostate tissue and inserted the entire normal prostate tissue cDNA library into VSV. They were then able to infect mice that suffered with prostate cancer and observe what happened to their tumours - specifcally, was an effective immune response generated and did the tumour shrink? VSV virus particles were injected into the mice, virus entered the cells of the mice and began to replicate and express their genes, including the newly inserted prostate cDNA. Essentially, thousands of virus particles were adminsired, each containing a slightly different gene from the prostate cDNA library. High levels of tumour associated antigens were therfore being expressed in mice allowing for the generation of an effective immune response.

Survival of mice treated with the VSV viruses - GFP expressing negative control; and the VSV ASEL in mice with established 'TC2' prostate tumours.

This approached effectively cured the mice who suffered from prostate cancer. Following this treatment a number of resistant tumours emerged which were again subjected to a further treatment using a cDNA library taken from the tumour itself this time and this readily treated the secondary resistant cancers. The clinical benefits of this approach can hardly go unnoticed. The ability to administer a broad tailored therapy that has the potential to cure an established tumour will be revolutionary, especially given the relative ease at which this can be developed 'at the bedside'. The ability to easily genetically manipulate viruses has - and will continue to - revolutionise the medical sciences. Look out for the eminent clinical trials. 


Kottke, T., Errington, F., Pulido, J., Galivo, F., Thompson, J., Wongthida, P., Diaz, R., Chong, H., Ilett, E., Chester, J., Pandha, H., Harrington, K., Selby, P., Melcher, A., & Vile, R. (2011). Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors Nature Medicine DOI: 10.1038/nm.2390

Improving global immunisation with more heat tolerant vaccines

During the last century, mass vaccination campaigns were rolled out across much of the industrialised world. And, with much success, we have near eradicated most common virus infections from these populations.   Despite the more recent efforts to bring the developing world into this fold, major setbacks have been uncovered, one being: how exactly are we to stably transport  delicate vaccine stocks from their place of production to the regions that need it most.

   Many of these vaccines, known as 'live attenuated vaccines' - or LAVs-are generally unstable in the environment outside of our cells and bodies. For example, the measles virus has an outer lipid membrane that is highly delicate and damage to this removes any chance of this vaccine working. Basically, these LAVs, which rely upon a weaker infection, cannot successfully complete their replication cycle without an envelope. Generally, the ability of a vaccine to provide protection is intrinsically linked to its overall structure; you destroy the structure, you destroy protection.

Unstable measles virus particles

  The more difficult places to transport vaccines to  happen to be more tropical regions, like sub-Saharan Africa, South America and South-East Asia. These places are very warm and humid which goes against what our vaccines like. With temperatures up to 40 degrees Celsius and the fact that freezing also destroys the vaccine, the only way forward is the implementation of a cold chain. If we keep the vaccine stock 'chilled' from manufacture (see Merck or the Serum Institute of India) all the way to administration, then we have a chance to prevent its degradation. But this is not as easy as it may sound as it is economically and logistically difficult to achieve this in developing countries. To overcome this, the produced vaccine stock is freeze-dried to remove any water that may contribute to its instability and when this prep reaches the clinic it is then reconstituted through the addition of a solvent preparation, which has then all got to be kept cool.

With measles virus vaccines however, even upon reconstitution, there are still major losses to immunogenicity and this problem is compounded with the use of multi-dose vials of vaccines. A single vaccine stock, prepared in the morning may now sit in the clinic unrefrigerated until the end of clinic hours. If we were then to get immunized at 5:00 pm, what are the chances of that being successful? It is thus not much of a surprise to hear that many virus outbreaks have been attributed to breaks in the cold chain like this. Our ability to eradicate many diseases in these countries and hence worldwide, is blocked by difficulties in the cold chain.

   Being so, Bill and Melinda Gates identified "Vaccines That Do Not Require Refrigeration" as one of their funded 14 Grand Challenges in Global Health and the results of one potential solution to this problem have just been published in the journal Vaccine (read it here). This work aimed at identifying novel reconstitution liquids that would increase the thermostability of the prepared vaccine and therefore increase its 'shelf-life' in these more difficult environments. Using a recombinant measles vaccine virus that expresses green fluorescent protein (GFP) upon infection, the group were able to assess the level of infectivity of a range of vaccine stocks reconstituted in varied solutions.

Counting measles virus infected (GFP positive) cells

   Indeed, they tested >11,000 formulations (myriad combinations of buffers, stabilizers, solubilizers, preservatives, pH and tonicifiers - whatever they are?) using their recently developed high-throughput system in which they were able to automate the whole process. These results were validated and confirmed with another virus, this time an adenovirus expressing GFP; results were the same. The group identified a formulation that caused the vaccine to "suffer <1.0 log loss after 8 h at 40 ◦ C in the liquid state", a major improvement on a previous loss of" 1 log of potency after 8 h at 37 ◦ C in the reconstituted (liquid) form". In conclusion, they identified a novel vaccine formulation that substantially increased the stability of two viruses, which can be used in immunization campaigns worldwide. As I've alluded to before, the use of high-throughput screens has much potential in the world of virus research.

Log loss of infectivity of vaccines

   How is this result likely to change vaccine implementation in the real world then? Well, the evidence presented here indicates the ability of creating more stable vaccines that would transfer to better vaccine coverage yet the real problem appears to be whether current vaccine manufacturers would accommodate such a significant change to production, especially given their inherent unwillingness to invest in the developing world.

   The cost associated with this may be offset by the savings made through reduction in vaccine wastage and loss of cold chain implementation but we must realise that only when such an improvement looks attractive to the market will change come. The future of vaccination looks to be bright, especially with the combination of newer technologies and the backing of large philanthropic organisations.

Schlehuber LD, McFadyen IJ, Shu Y, Carignan J, Duprex WP, Forsyth WR, Ho JH, Kitsos CM, Lee GY, Levinson DA, Lucier SC, Moore CB, Nguyen NT, Ramos J, Weinstock BA, Zhang J, Monagle JA, Gardner CR, & Alvarez JC (2011). Towards ambient temperature-stable vaccines: The identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay. Vaccine PMID: 21616113

Can we target antivirals to the host cell?

Two papers published recently explore the idea of targeting antiviral compounds to the host cell using high-throughput organic synthesis and an in vitro screen. Both groups identify a single novel inhibitor of virus replication and both are host specific.  One paper goes further and identifies the target and also the in vivo potential of this compound. These papers highlight the hopes and pitfalls of targeting the host cell against viruses while shedding light on the basic biology of these viruses.

   We can protect ourselves from the harmful effects of viral infection through the use of vaccination, antivirals or indeed behavioral changes. Yet not all viruses have proved to be amenable to these and some, like measles and mumps, are quite easily combated with mass immunization campaigns while the likes of HIV have shown to be quite sensitive to antiviral compounds.

Respiratory infections are a problem! hastings.gov.uk

   For a majority of viruses though, we have neither effective vaccines nor antivirals. We need these now more than ever. And, even those which we do have vaccines and/or antivirals (Influenza A), resistance is quickly built up over time rendering them clinically useless. How then are we able to generate new, more effective antiviral molecules against a whole range of viral pathogens, especially given the cost of taking a single drug to patients?; we want more bang for our buck with antivirals. One way maybe through the targeting not of a viral gene or protein but a cellular one. The virus life cycle is so intimately connected to that of the host cell that removal of cellular pathways may have a dramatic effect on virus functioning and applications like these have the beauty of possibly targeting both established and emerging pathogens.

   Krumm et al, from the Emory University School of Medicine, Atlanta, U.S.A, demonstrate the feasibility of this approach using viruses from the paramyxo- and the orthomyxoviruses (generally called the 'myxoviruses') as their model of choice. Read the paper here. Using this, they discover a novel compound that demonstrates host-cell specificity while greatly inhibiting virus replication. Even at nanomolar concentrations (potent!).

   This work falls on the back of a series of experiments they reported back in 2008 where they screened a 137,500 compound library against in vitro infection with the related measles, canine distemper and human parainfluenza 3 viruses. This was to determine if they could uncover broad range, potent inhibitors of a large number of viruses. Moving away from the 'one-bug, one-drug' paradigm, this work identified a class of 11 compounds that did just that: they significantly inhibited replication and cytopathic effect of all the three viruses. They use the data from this series of experiments to find a host-targeted compound.

They state that their 'ideal' inhibitor would be:

In search of candidates with a host-directed antiviral profile, we anticipated three distinct features of desirable compounds: a) potent inhibition of virus replication at the screening concentration (3.3 µM); b) a primary screening score, representative of the selectivity index (CC₅₀/EC₅₀), close to the cut-off value for hit candidates due to some anticipated host-cell interference ( = 1.9); and c) a broadened viral target spectrum in counter screening assays that extends to other pathogens of the myxovirus families

initial compound A) and B), the synthetically optimised JMN3-003

   A single compound was taken forward and then synthetically optimized (high-throughput synthetic chemistry anybody?) to achieve even better inhibition. No longer do we have to rely on the natural diversity of compounds out there. This new and improved compound (JMN3-003) could inhibit not just myxo-virus replication but also that of clinically relevant RNA (Sindbis virus) and to a lesser extent DNA viruses, such as vaccinia virus. The broad activity of the molecule suggests that it may target a more general aspect of viral replication than others had previously. The activity of this drug was also host-cell specific, suggesting that it did not target a virus structure but rather one found within the host cell, whose activity would change depending on the species assayed.

JMN3-003 antiviral activity

   This molecule had little or no effect on the metabolic activity of the immortalized cell lines used or of other primary cell cultures when administered at >7,000X the active dose, possibly hinting at the safety profile of such a drug. And, under liver cell extract stability tests, the compound showed a desirable extrapolated half-life of around 200 minutes. Host transcription and translation weren't affected by drug administration.To be safe, the authors put forward that this compound may only be preferentially used in acute respiratory infections where the treatment time would be substantially shorter. This group did not carry out any in vivo work so the real safety of this compound cannot really be assessed. See below for how this should be done.

Activity changes depending on the cell used. Human PBMCs or primate Veros

   So, we have demonstrated that the compound inhibits virus replication. But just how exactly does it do this? At what stage of the virus replication cycle does it act? Entry? Transcription? replication? assembly? exit? The group addressed these points through the use of assays that measure virus envelope fusion with the cell membrane (virus entry) and virus gene expression and replication (post entry). This work showed that fusion was not inhibited when the drug was added suggesting a post entry step was being targeted. When compared to a known inhibitor of virus polymerase activity, JMN3-003 looked very similar. Other experiments, assessing levels of mRNA and genomes produced from virus replication showed a significant inhibition in one or both processes during measles or influenza virus infection. Some component associated with virus polymerase reactions is being targeted. What it is, sadly this paper did not address, yet further experiments I am sure will uncover the mechanism of action of this compound and maybe through this identify an important host-virus association that has been previously overlooked. Recently, evidence has suggested that these viruses require the activity of host kinases for proper, regulated replication. Could it be these?

Little risk of resistance?

   Their experiments also suggest that it is extremely difficult for these viruses to adapt to the inhibition from JMN3-003. After 90 days of replication on cells treated with the compound, little resistance had emerged. Again, the reasons for this were not addressed. When compared to previous problems of drug resistance seen in Influenza A and HIV-1, this activity looks very promising.

RSV - the biggest cause of bronchiolotis in young children. http://www.empowher.com/

   Interestingly, this approach has been used for another medically important virus (Respiratory syncitial virus, RSV - also a paramyxovirus) and just published recently in the journal PNAS (Bonavia et al 2011). This group used effectively the same strategy as described above, except using a lot more compounds (1.7 million). They picked up a number of molecules that also targeted a post-entry step in virus replication, possibly virus replication ( RdRp activity); it also shows broad spectrum activity against a range of RNA viruses. And again, no resistant viruses emerged in the experiments. 

   This work went a little further than that I mentioned earlier and Bonavia et al were able to pull down the drug target using a type of affinity purification with immobilized samples of the compound (iTRAQ quantitative chemical proteomics). The protein binding most strongly to this may mostly likely be the target. In this case, the two compounds tested both targeted seperated enzymes in the same pathway, the de novo pyrimidine biosynthesis pathway, that is the nucleotides A, T and U (A and U being used extensively by RNA viruses). To assess the safety of targeting this important cellular pathway, the group looked at both in vitro and in vivo rodent models of disease. Rapidly-dividing cells showed high sensitivity to the compound while significant histopathological changes were observed in lymphoid and gut tissues in the cotton rat. This is not good for a potential clinical drug that may be administered to very young babies. 

Pyramidine biosynthesis - a good antiviral target? http://upload.wikimedia.org/

   Sadly, these compounds exhibited no antiviral activity in vivo and rather one increased RSV virus replication. This is possibly down to the potential immunosupressive nature of the target (inhibits rapidly dividing cells - T and B cells) or the ability of circulating pyramidines in the animal's body to rescue the inhibition of their synthesis inside the cell.

   I wonder how the first compound, JMN3-003 would fare in the many animal models of myxovirus infection. And, what would the target be when the chemical pull-down was applied? would it be the same pathway? Both these papers highlight the ability of this approach to identify readily targeted host cell components to reduce virus replication in vitro and potentially overcome the problems of narrow virus targeting and resistance while the second paper goes further and shows us how the results may not be pleasing. The targeting of host factors has obvious implications for patient health and this cotton rat model displays what can go wrong. We should learn from this and expand any potential drug candidates into in vivo modeling quickly and at the start of any experiments, not like what was done with JMN3-003, although that's not to say the same will happen with that compound.



Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K., Monroe, J., Wolff, K., Osborne, C., Lanieri, L., Hoffmaster, K., Amin, J., Markovits, J., Broome, M., Skuba, E., Cornella-Taracido, I., Joberty, G., Bouwmeester, T., Hamann, L., Tallarico, J., Tommasi, R., Compton, T., & Bushell, S. (2011). Organic Synthesis Toward Small-Molecule Probes and Drugs Special Feature: Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV) Proceedings of the National Academy of Sciences, 108 (17), 6739-6744 DOI: 10.1073/pnas.1017142108

Krumm, S., Ndungu, J., Yoon, J., Dochow, M., Sun, A., Natchus, M., Snyder, J., & Plemper, R. (2011). Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases PLoS ONE, 6 (5) DOI: 10.1371/journal.pone.0020069

Yoon, J., Chawla, D., Paal, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J., & Plemper, R. (2008). High-Throughput Screening--Based Identification of Paramyxovirus Inhibitors Journal of Biomolecular Screening, 13 (7), 591-608 DOI: 10.1177/1087057108321089