Merck's mumps vaccine is good. But not good enough.

It came out this week that two former employees are suing Merck (here's the document), the pharmaceutical company/giant, over what they say was a deliberate attempt to con the US government - and by extension many other countries - into buying a mumps vaccine in the form of their combination MMR-II vaccine that was not as effective as they had previously stated. 


As documented here this has been jumped on by a number of anti-vaccinationists but if you really look at the data from the "real world", you'll quickly see how all parties have really jumped the gun a bit on this issue and that clearly the vaccine is - and has been - working well. But it could be better.

Single mumps vaccine jab (versus MMR) in the UK - back again?

The "Children's Immunisation Centre" has recently announced that it plans to start administering the single mumps vaccine sometime in the 2nd quarter of this year in the UK. When I rang them up asked at the helpdesk, the very helpful receptionist told me they were scheduling vaccinations for July. Their website details:

"Currently Mumps single vaccine is not available due to Merck stopping the manufacture of it about 3 years ago. However we are working with our suppliers (who have an MHRA -Medicines and  Healthcare products Regulatory Agency - Wholesale license and who are regularly inspected) to source a Jeryl Lynn Strain (the safest strain) of Mumps vaccine." 

They continue,

"M-M-R single vaccines are manufactured by, France, India, and in some European countries and also China, Russia and in future the  UK. Our suppliers in the UK are working with UK manufacturers and are looking to produce the single mumps vaccine  for our patients."

The internet has picked up on this as well, here.

More UK mumps outbreaks in Glasgow, Scotland.

Typical mumps swelling.

I've written about this a lot - which isn't all that surprising given that our lab is one of a handful working on the virus - but mumps has struck again, this time raising it's head in Scotland. Here's a nice article.

The University of Glasgow has experienced more than the usual (a handful probably) of cases of mumps infection. Currently 15 students have been diagnosed with the virus which usually causes swollen salivary glands under the neck, although a number of more serious symptoms can result. The University is working closely with the community control the outbreak and to vaccinate those unprotected.

HIV restriction factor blocks respiratory viruses - but not how we thought

Measles virus - is it targeted by an HIV restriction factor?

Cellular organisms have evolved multiple defences to keep viruses and other genetic parasites at bay. One such shield is the development of an advanced adaptive immune system seen in vertebrates while another is the more evolutionary widespread 'innate' system made up of various expressed proteins and small RNAs. These molecules prevent particular stages of the viral replication cycle like entry, replication or exit. 

Another mechanism is to mutate the virus out of existence, i.e. change nucleotides throughout the viral genome so much so as to effectively knock-out that protein's function during infection and slow down replication so much so to allow your bodies' other defences to clear it. Since 2002 we have had tantalising clues that this is functioning during a viral infection, particular HIV and other retroviruses:

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.

Influenza - putting the Trojan into the horse but should you open it?

A trojan horse (dendritic cell) filled with virus

Inspired by a recent journal club article:

A number of pathogens infect via one organ but are able to move to another. Think of the likes of the initial HIV infection of cells within the reproductive tract followed by its transfer to the immune system (see my earlier post here). This transfer of viruses, bacteria or other parasites is responsible for the induction of an effective immune response but also it can lead to some of the more serious disease symptoms during infection, but how is it controlled?

Are microbes evolving to escape vaccination?

What if all those vaccines - those ones that work really well - all stopped working? Imagine if the viruses and bacteria from which they are trying to protect you against, evolved and adapted to life in a largely immune population? Those robust antibody and T cell responses generated within a person following vaccination supply the perfect breeding ground for the selection of resistant mutants where anibodies can no longer recognise and neutralize their targets and where T cells fail to eliminate infected cells. So, is it possible and is it happening?

Well, we already know this kind of phenomenon from influenza, right? Every year we have to change the strains that are put into your flu jab to match those viruses predicted to be circulating come winter. This is based on generating an antigenic match of vaccine to wild virus; specifically, their surface HA proteins must look the same. This is why there has been such a push to develop universal influenza vaccines capable of immunizing people against all flu strains. For viruses like measles and mumps however, we have our universal vaccine, or at least we thought we did.

Influenza may change through antigenic drift and shift forcing us to develop new vaccines each year, but do other viruses evolve through antigenic drift and force us to generate improved vaccines for them? http://news.bbc.co.uk/

Mumps outbreaks - why here, why now? and how can we stop it?

Mumps is an acute viral disease of humans and really only humans. It is spread through the respiratory tract (probably droplets and aerosols) where the virus most likely infects the cells lining the epithelium - we don't really know this for certain. From here it spreads throughout the body, infecting organs and tissues such as the salivary glands, testes and ovaries, pancreas, and perhaps most worryingly, your central nervous system. In fact, in the pre-vaccine era, mumps was the biggest cause of aseptic meningitis across the developed world; it can also cause the more severe encephalitis.

Lucky we've got a vaccine, right?

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

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

So, how do you know when a vaccine is safe?

How can you tell how safe a vaccine is?

Mumps, a highly infectious viral disease, has been largely eradicated in the developed world following the introduction of a highly effective live-attenuated vaccine. Highlighted by well-publicized outbreaks in the U.S and U.K, the number of cases, however, has risen causing worldwide alarm. The reasons for this re-emergence have yet to be fully elucidated but most likely are due to a number of factors, including waning immunity and poor vaccine coverage.

Despite what is normally reported, mumps infection can cause serious complications. Prior to the introduction of the vaccine - and of course in countries that fail to administer it - mumps infection was/is the most common cause of viral meningitis and encephalitis; it has been estimated that 50% of those infected by mumps have some form of central nervous system involvement, although 1 - 10% will actually experience a symptomatic infection. It is safe to say that the mumps virus is one of the most neurotropic human viruses currently circulating and that its neurotropism can hardly be considered a complication.

All this really underlines the importance of maintaining mumps vaccination in protecting individuals and populations from serious disease. The key then is to develop not just more effective vaccines but also safer vaccines as people aren't likely to give their children a vaccine which may cause serious side-effects especially considering the propensity for mumps virus to cause CNS disease. A recent review of mumps vaccine safety states that,

Such a problem places public confidence in all mumps vaccines at risk, as indicated by the experience in Japan where national mumps vaccination programs were discontinued in 1993 following established links to aseptic meningitis; consequently, more than a million new mumps cases occur annually in that country

Lewis rat - is this the future of mumps vaccine safety?

How then are we to assess the safety and more specifically 'neurovirulence' of mumps candidate vaccine stocks? Recently, Rubin and Afzal from the United States Food and Drug Administration and the UK National Institute for Biological Standards and Control respectively, outlines the current state of the art in mumps virus safety testing and outlines how its future might look. What we would like in a test system is for it be accurate and fully predictive (limit false positives and negatives); it would need to economical (vaccines need a lot of testing) and it needs to be relatively easy to carry out and replicate. For us to do this, these methods require vigorous testing!

Currently, much like other virus vaccines, mumps vaccine safety is assessed in a monkey model and has resulted in the detection of significantly attenuated vaccines for use in humans. There is however cause for concern with this system as in some instances it fails to distinguish between important differences in levels of attenuation. There is therefore a need to replace this system if not on the grounds of ethical and economic concern but on the grounds of safety. In has stepped a small animal model - the lewis rat- which has been shown to better predict neurovirulence; is cheaper and is less ethically taxing; it is hence subject to a WHO validation study.

False colour electron micrograph of the mumps virus

But why do we have to use animal models at all for safety testing? Can we not just be content with in vitro studies with cell lines? In some cases, we can predict how a virus will act within an animal on the basis of studying how it infects and replicates in cell line but there is, however, no in vitro alternatives for mumps - at least not yet - and even if there were we can't say whether it could ever fully replace animal studies.

In some systems, animal infections just cannot be replaced if we are to maintain a high level of vaccine safety which of course is important when vaccines are administered to billions of people worldwide we are then forced to stick with animal testing. We can rest assured that with recent developments in small-animal models, future testing may come more accurate, cheaper and a little more ethically pleasing.

BRUYN HB, SEXTON HM, & BRAINERD HD (1957). Mumps meningoencephalitis; a clinical review of 119 cases with one death. California medicine, 86 (3), 153-60 PMID: 13404512

Dayan GH, & Rubin S (2008). Mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 47 (11), 1458-67 PMID: 18959494

Rubin, S., & Afzal, M. (2011). Neurovirulence safety testing of mumps vaccines—Historical perspective and current status Vaccine DOI: 10.1016/j.vaccine.2011.02.005