Do we really need a new measles vaccine?

Measles, that deadly childhood infectious disease is almost a distant memory to most people nowadays, that is except for a few isolated outbreaks across the US and Europe. This is all because of a really amazing preventative therapy: the vaccine.

Vaccines are great. They are by far the most effective means that we have to control - and hopefully eradicate - infectious diseases from a range of species. Measles is one of these diseases that, over the last half a decade or so, we have backed into a corner across the world. Before the introduction of global immunisation the measles virus caused around 2.6 million deaths, most of which were children. To show just how great the vaccine is: 2008 saw only 164,000 deaths (see the WHO data here). A big number still but a 97% decrease in associated fatalities is pretty impressive, so why then - in an editorial piece in the esteemed journal Vaccine, are they calling for researchers to develop a new vaccine?

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?

Revisiting the origins of contagion - the measles story

The mechanisms behind the incredible infectiousness of measles are poorly understood - that is, until now, where two studies have now come forward investigating the molecular biology of measles person-person transmission. Two groups have independently identified the protein, nectin-4 (a cell adhesion molecule) as the receptor allowing measles to infect and emerge from the respiratory tract and spread from person to person, potentially filling in a major gap in our understanding of this important human pathogen.

The measles virus is one of - if not the most - infectious agents currently circulating in human populations. It is also responsible for considerable disease and death worldwide, particularly across the developing world. But luckily, the introduction of a live-attenuated vaccine has significantly reduced it's incidence - this is in spite of a number of recent outbreaks associated with reduced vaccination rates. 

Did dinosaurs get measles?

 I know what you're thinking: of course they did - what organism out there isn't parasitized by viruses and their ilk? But where's the evidence? In science you can't just pluck ideas out of the air like this - no matter how obvious they are - and say they are true; maybe dinosaurs had a fantastic immune system and were able to thwart any potential viral invaders. What I'm saying is that we just don't know, but a paper published last month in Current Biology suggests otherwise: documenting the discovery that some dinosaur (see above) fossils bare lesions in their bones that are suggestive of viral infection.

Closing the book on measles infection - do we know it all?

Measles infection. (http://biowiki.org)

One of most important factors in establishing a viral infection is the presence or absence of particular receptor molecules. These proteins/sugar molecules (or whatever) must be expressed on the surface of the cell, allowing the virus to bind and initiate entry. 

The expression of a receptor therefore governs the behavior of an infection by allowing only certain cells to permit virus replication. This thus controls how a virus enters your body, spreads throughout it, causes disease and finally escapes to continue the infection in a new host. The identity of the receptor that a virus uses allows a better understanding of infection and pathogenesis and may facilitate the development of new antiviral treatments.

A paper published in PLoS Pathogens (see here), this week documents the discovery that a protein - Nectin-4 - acts as the receptor that may allow measles virus (MeV) to escape your body in the ends-stages of the disease, through the infection of the epithelial cells lining your respiratory tract. However, this molecule differs from that used bythe virus to initially enter your body.

This Nectin-4 study finally sheds light on what has been an elusive molecule to find and goes a long way in explaining the basic biology of this historic human pathogen. Also, the findings that Nectin-4 is highly expressed on the surface of a number of common cancers further adds weight to the use of MeV as an anti-cancer agent. But does Nectin-4 fully explain MeV infection? And, can we finally close the book on measles infection?

Despite the widespread use of a highly effective - and safe - vaccine, MeV still infects an estimated 20 million people a year, mostly young children and, in 2008, the WHO documented that measles caused the death of 164,000 of these children that year. Measles is characterised by fever, photophobia, cough, runny nose, sickness, and a nasty rash over most of the body (see picture above). Every so often the virus will enter your brain and can cause serious inflammation there. And, even more rarely will the virus persist in your body only to re-emerge years later as an incurable degenerative brain disease known as sub-acute sclerosing pan-encephalitis (SSPE). The molecular basis of how this virus enters our body, replicates, spreads inside you exits your body are only now being realised with modern recombinant viruses, like those expressing fluorescent proteins (see below). For examples, see here, here, here and here. 

Disclaimer: these references are from work that my supervisor and the group I am currently in are heavily involved in but are the only work documenting MeV in the entire organism. Read here when I previously wrote about the discovery of how MeV enters our body through dendritic cells.

GFP-expressing MeV infection in a Rhesus macaque model - many organs afflicted and recapitulates human disease. Virus replication in, A) Outer skin, corresponding to rash, B) oral mucosa, C) tongue and tonsil, D) lymph node, E) Lung, F) stomach and intestine (gut-associated lymphoid tissue - GALT), G) intestine and spleen, H) and I) both close-up of 'koplik spot' skin rash. (de Swart, et al 2007)

It was always assumed that following breathing in virus-laden aerosols MeV initially infected your epithelial cells lining the upper respiratory tract. From here it could spread throughout your body, replicating in lymphocytes and finally be released back out via the epithelial cells. But, researchers using a green-fluorescent protein (GFP)-expressing MeV showed that it was in fact dendritic cells (sentinal immune cells) found within your respiratory tract that were the first cells to be infected. These cells could then easily transmit the virus to your immune cells. These cell types can be justified through the identify of the receptor for measles, SLAM that is only expressed on these immune cells. 

GFP-expressing MeV infection of A) polarised epithelial cells, B) and C) lymphoid like cells in mucosa. (de Swart et al, 2007)

However, based on animal models and autopsy reports, we know that the virus can - and does - productively infect epithelial cells lining your airway (see figure above) yet no receptor was known. That is, until Chris Richardson's team at Dalhousie University, Canada discovered that Nectin-4 could function as a MeV epithelial receptor. 

PVRL4 (Nectin-4) allows MeV infection

The group used microarray analysis to determine what genes were expressed in cells that could, and those that could not, be infected by a wild-type, pathogenic MeV. 

They were then able to bioinformatically pull out proteins that were found on the plasma membrane surface (ones that were likely to be receptors) and expressed them in cells that couldn't be infected. The gene that allowed MeV to infect was the receptor molecule and this was confirmed to be Nectin-4 through siRNA knockdown and antibody binding assays.

So does Nectin-4 explain how MeV infects, causes disease and escapes the human body? Based on the Human Protein Atlas entry for Nectin-4, this protein is on most normal cells weakly - including the brain and lung, which are both targets for MeV and it is not really expressed on lymphoid cells, although these cells already express a highly-active receptor for the virus. This study may explain how the virus can infect your respiratory tract epithelium and be secreted into your airways to spread the infection. The role of Nectin-4 should be established in further model systems like the macaque and even in vitro differentiated epithelial cells.

de Swart RL, Ludlow M, de Witte L, Yanagi Y, van Amerongen G, McQuaid S, Yüksel S, Geijtenbeek TB, Duprex WP, & Osterhaus AD (2007). Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques. PLoS pathogens, 3 (11) PMID: 18020706 


Lemon K, de Vries RD, Mesman AW, McQuaid S, van Amerongen G, Yüksel S, Ludlow M, Rennick LJ, Kuiken T, Rima BK, Geijtenbeek TB, Osterhaus AD, Duprex WP, & de Swart RL (2011). Early target cells of measles virus after aerosol infection of non-human primates. PLoS pathogens, 7 (1) PMID: 21304593

Noyce, R., Bondre, D., Ha, M., Lin, L., Sisson, G., Tsao, M., & Richardson, C. (2011). Tumor Cell Marker PVRL4 (Nectin 4) Is an Epithelial Cell Receptor for Measles Virus PLoS Pathogens, 7 (8) DOI: 10.1371/journal.ppat.1002240

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

The Grand Challenge of Aerosolised Vaccines

Despite the development of effective vaccines, many human populations are currently at the mercy of numerous endemic viral pathogens. Measles virus is one such pathogen that, in 2008, was responsible for 164,000 deaths; the worst effected areas are South-East Asia and Africa (WHO stats can be found here). You might find this surprising as there is currently a very good measles vaccine in use – in fact you probably received at some point during childhood and are protected from future infection. Measles cases have been significantly reduced in the developed world, so why hasn’t this vaccine allowed for the eradication of measles virus transmission in the developing world?

Needle vaccination against measles

The key to controlling measles – and other viruses – is to generate sustained high levels of good quality immunity within a population so that the virus can no longer successfully infect and has nowhere to go; this is known as herd-immunity. The problem then is, well why can’t we achieve the herd immunity required to prevent virus transmission? In places like Africa, where people are reminded daily of the horror of measles, you don’t have to force them to accept vaccination unlike what was seen in the UK and US recently so they are readily vaccinated. One problem however, appears to be the mode of vaccination, that is injecting the virus vaccine creates hurdles to a successful immunisation campaign:

·      Trained healthcare workers are required to safely administer the vaccine when it is injected

·      The currently used vaccine formulation tends to go off in temperatures ~ 37 degrees Celsius causing problems for transport and storage especially in areas such as Africa.

·      The use of used/contaminated needles may facilitate the problems of blood-borne diseases and drug use

Are there any alternatives to needle vaccination?

There are of course other ways to vaccinate people, maybe the respiratory tract or the gastrointestinal tract may make better options – especially considering how different injecting a virus is to most of their natural entry mechanism. The mucosa-associated lymphoid tissue, lining the mucosal epithelium of thegastrointestinal and respiratory tracts may also prove to be a more effective place to induce stronger immune responses.

Currently, some 3million children have already been successfully vaccinated from measles using a ‘wet’ aerosol delivery system, however the formulation was unstable above 4 degrees Celsius and delivery was difficult. A recent paper published in PNAS has sought to improve upon this current measles vaccination technology (and also get around the problems of injecting vaccines) through the generation of a highly immunogenic respiratory-delivered ‘dry’ vaccine formulation. It was tested in macaques, can be administered as a single dose and is a highly thermostable, powdered formulation.

'PuffHaler' aerosol delivery system for 'dry' vaccines

 So, how good is it?

They report that their aerosol-delivered vaccines were deposited into the upper and lower respiratory tracts and resulted in the generation of good-quality measles virus specific humoral (B cell + antibody) and cellular (CD8+/CD4+ T cells) immune responses without safety concerns; there also exists a long-lived (<1 year) B cell memory function (and some T cell memory) correlating with long-term virus protection. They show the their vaccine strategy allows for the successful protection from subsequent measles virus challenge. Through comparisons with injected vaccine, the group were able to show that indeed all routes of vaccination generated the required level and quality of immunity required to protect from measles but given other concerns with injecting vaccine aerosol delivery may prove better. These results indicate that this proof of concept, novel vaccine may be comparable to the previously used formulation – although human studies would have to be carried out.

Aersoal delivery system

Scientists are being allowed to investigate problems like these only through being funded by the 'Grand Challenge in Global Health Grants' via the Bill and Melinda Gates Foundation; the money supplied allows for the development of better, safer and ultimatey more cost-effective vaccines. This work highlights the importance in the development of these new and more effective vaccine technologies in order to facilitate the eradication of viral pathogens worldwide. How might this method of administration affect other vaccines, only further work will decide but if we are unable to prevent measles transmission with a highly effective vaccine then what hope do we have to prevent other, less well-studied viruses without decent vaccines?

Lin, W., Griffin, D., Rota, P., Papania, M., Cape, S., Bennett, D., Quinn, B., Sievers, R., Shermer, C., Powell, K., Adams, R., Godin, S., & Winston, S. (2011). Successful respiratory immunization with dry powder live-attenuated measles virus vaccine in rhesus macaques Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1017334108

HIV & Measles - double hit pathogenesis?

Despite ongoing worldwide eradication efforts, measles infection still results in significant morbidity and mortality. Although, throughout most of the developed world measles infection has been considerably reduced there still exists massive (and deadly) outbreaks in areas such as Africa and South-East Asia. Investigation of the reasons why this disparity occurs therefore  is of major medical, political and social interest.

Many factors are likely to be behind this major difference - and all of which deserve our attention if we are ever to remove measles from the human population. There exists problems in rolling out vaccines in countries with poor infrastructure such as roads and transport facilities; disruption to what is known as the vaccine 'cold-chain' (vaccines have to be kept cold to avoid rendering them unusable) is likely to occur; general poor health of the population in these regions and possible interference of vaccination in children with high levels of passively acquired maternal antibody.

Measles vaccination efforts in Africa may not be entirely effective

Today in PLoS Pathogens, Nilsson and Chiodi highlight in a featured opinion article, another possible source: the link between co-infection with HIV-1 and Measles infection. They point out that HIV-1 infection and replication may result in impaired immune responses in both mothers and children leaving open the possibility of measles infection (no immune system, no protection). HIV-1, as I'm sure you will all know, is a potentially deadly pandemic retrovirus - particularly a major problem in sub-Saharan Africa- which infects humans where it resides in the bodies own immune system: T cells, dendritic cells and macrophages. Viral replication results in the death of these immune cells and destruction of important lymphoid tissues resulting in an individual without key immune functions.

The authors note that children born to mothers who are HIV-1 positive or are HIV-1 positive themselves develop lower levels of anti-measles antibody upon vaccination -a big deal if we're looking to protect these kids through vaccination. They show that memory B cells may be impaired and lower protection will result through failure to mount a B cell-generated antibody response. Immunity is a highly regulated system, if you remove one aspect-  in this case T cells - you will affect another pathway , in this case B cells. Thus there exists a major  problem with HIV-1 infected people and infection with other pathogens in the environment; HIV-1 infection significantly alters the host immune system weakening it to other invading pathogens such as measles which is endemic in these areas.

So how do we overcome this problem? Well, the authors suggest that on top of increasing vaccination coverage through catch-up programs it would be wise to administer anti-retroviral drugs  to mothers and children prior to vaccination to allow sufficient immune function; this should hopefully make a difference in combating both measles and HIV in the developing world, especially in an area where both cause so much pain. Hopefully, strategies such as this will aid treatment efforts for other pathogens rife in the developing world - targeting both HIV and the individual agents may be more effective.

Sadly, there exists another interaction between HIV and co-infection with other pathogens. Infection usually results in increased levels of immune cells in the blood and tissues yet these very cells are the target for HIV and if these cells increase, HIV replication will also. There exists a deadly interaction between multiple pathogens which must be broken.

Nilsson, A., & Chiodi, F. (2011). Measles Outbreak in Africa—Is There a Link to the HIV-1 Epidemic? PLoS Pathogens, 7 (2) DOI: 10.1371/journal.ppat.1001241

Can fluorescent-‘labelled’ viruses illuminate their mechanisms of pathogenesis?

Have you ever wanted to visualise viral infection? Ever wanted to observe how they enter and spread throughout their host organism? Ever wanted to know how exactly they caused disease - at the cellular and whole-organism level? Well, this may be entirely possible using fluorescent-labeled recombinant viruses infecting a relevant model system.

[caption id="" align="aligncenter" width="504" caption="GFP-virus infected cells"][/caption]

So how does it work?

Lemon et al recently report the continued investigation of measles virus pathogenesis in a non-human primate (Macaque) model utilising a green-fluorescent protein (GFP) expressing virus. Upon infection of host cells, viral transcription leads to the very high expression of GFP, flooding the cytoplasm with this fluorescent ‘tag’. Subsequent microscopy, imaging and immunohistochemistry allows for the identification and location of the infected cells, tissues and organs - see image above. Tracking of cellular infection allows us to decipher the development of MeV entry, spread and replication at both the cellular and whole-organism level throughout the entire infection. Studies such as these give an unprecedented view of viral infection in a means directed related to that of human infection. This model even allows for macroscopic real-time detection of fluorescence and hence viral infection.

Why is this important for measles?

Despite a highly effective vaccine and significant global control initiatives, measles infection still accounts for significant morbidity and mortality worldwide, mostly in the developing world (164,00 deaths in 2008). This is mostly attributable to the profound immunosuppression induced allowing for further infection with opportunistic pathogens. Currently, much is known about measles pathogenesis yet the molecular mechanisms of such are poorly understood and it is therefore of great interest to better understand these processes by which MeV infects and causes disease in humans. Knowledge of such may facilitate the development of more effective and safer vaccines for measles and indeed other viral pathogens.

Viruses being obligate intra-cellular parasites, must enter and exit cells in order to survive. Most of viral pathogenesis can therefore be attributed to the effects of viral replication of host cells and tissues; a major determinant of which is the expression of receptors on host cells surfaces allowing viral entry, infection and replication. Currently only a single receptor – CD150 - (otherwise known as signalling lymphocyte activation molecule SLAM) has been discovered that wild-type pathogenic MeV uses to enter host cells; the distribution of which only explains part of measles pathogenesis as epithelial and neuronal cells (important target cells) do not express the protein. As indicated by this receptor being expressed on lymphocytes and other immune cells, MeV is a highly lymphotropic virus! But if epithelial cells fail to express the receptor on their surface, how come its possible for MeV to enter via these cells?

The classical view of measles pathogenesis was that free-virus entered the host through the respiratory route, infecting and primarily replicating within the epithelial cell lining of the respiratory tract. Newly produced virus spreads to nearby lymph nodes where infected monocytes – a type of immune cell - facilitates viral dissemination throughout the host, resulting in the well-known symptoms of measles. The problem with this being that epithelial cells and unstimulated monocytes fail to express the MeV receptor CD150 and infection should therefore not occur. Recently, it has been shown (again using a GFP expressing virus in a macaque model) that MeV predominately infects dendritic cells during the peak of infection, ruling out a major role for monocytes. There is also however no direct evidence of MeV primary replication within the epithelium of the respiratory tract at the early stages of infection. So what exactly happens during the start of infection and does it develop? GFP-expressing viruses may shed light on this question.

[caption id="" align="aligncenter" width="415" caption="Diagramatic representation of the cellular composition of the human respiratory tract - notice the epithelial cell lining and the alveolar macrophages. Dendritic cells are however not shown on this diagram."][/caption]

So how can we study the early stages of infection?

The incubation period of  measles is about 2 weeks in humans making it particularly difficult to study the early events of viral infection – the kind of events like host entry, initial site of replication and subsequent intra-host dissemination - this is where we can use a non-human primate model.

Lemon et al  generated a highly virulent recombinant MeV based on viral isolates from an outbreak in Sudan; they engineered the viral genome so that it expressed GFP upon entry into cells – an addition that causes little or no replication defects to the virus. Groups of macaques were subsequently infected via the respiratory route allowing highly sensitive visualisation of GFP expressing cells following necropsy. The early time-points of around 5 days post infection were focussed on in this investigation allowing the determination of the early cell targets - epithelium? Immune cells?

So what did they find?

Their results suggest that at the early stages of MeV infection, GFP and hence viral replication is only found in immune cells within the respiratory tract and not the epithelial lining. Dendritic cells and alveolar macrophages are believed to capture viral particles in the lungs allowing spread via infected cells. This is known as a Trojan horse entry mechanism like that used by HIV to pass through mucosal tissues and infect humans - see below. This infection allows for spread and localised replication within nearby lymphoid tissues and then on to draining lymph nodes where massive lymphocyte cell infection may occur facilitating dissemination throughout the host, mainly within lymphoid tissues. Virus can be carried through host blood vessels to other lymphoid target tissues like the tonsils and adenoids and the gut-associated lymphoid tissue ' Peyer's patches'.

[caption id="" align="aligncenter" width="465" caption="HIV entry mechanisms utilising dendritic cells to pass through epithelial cell barriers - the 'Trojan horse' mechanism. This may be directly analogous to MeV entry and primary spread except in the respiratory tract."][/caption]

What does this mean?

This study clearly demonstrates the importance of non-epithelial cells such as dendritic cells in MeV entry, early replication and subsequent systemic spread. It does not however, rule out a major role for epithelial cells in later stages and in transmission - MeV still infects non-CD150 expressing cells and currently the mechanisms of which are unknown. Focusing on the later stages of infection may allow us to appreciate the other cell targets in pathogenesis and viral transmission. As mentioned previously, the use of fluorescent-labeled viruses offers an unprecedented view of viral entry, spread and pathogenic mechanisms. We should look forward to the time when studies like these are applied to other viral and indeed non-viral pathogens.

Lemon, K., de Vries, R., Mesman, A., McQuaid, S., van Amerongen, G., Yüksel, S., Ludlow, M., Rennick, L., Kuiken, T., Rima, B., Geijtenbeek, T., Osterhaus, A., Duprex, W., & de Swart, R. (2011). Early Target Cells of Measles Virus after Aerosol Infection of Non-Human Primates PLoS Pathogens, 7 (1) DOI: 10.1371/journal.ppat.1001263

Coombes, J., & Robey, E. (2010). Dynamic imaging of host–pathogen interactions in vivo Nature Reviews Immunology, 10 (5), 353-364 DOI: 10.1038/nri2746

Measles, Papua New Guinea and the brain

You may not have realised that - since most people nowadays have been vaccinated against it and have never seen it - but measles is a very serious illness. Generally an acute disease of children, measles is spread by the measles virus where it infects the body via the respiratory route and establishes a systemic infection - involving multiple organ systems - via your bodies own immune cells leading to the typical rash, mild to severe respiratory distress and immunosuppression (Rima and Duprex 2006).

[caption id="" align="aligncenter" width="463" caption="Measles virus replicative cycle"][/caption]

In the 'developed' world we tend not to think about infectious disease in the same way as people in other parts of the world; national vaccination campaigns have largely removed the threat (not considering some minor outbreaks) of the some of the biggest human killers and we no longer worry ourselves over whether a family member will come down with these diseases.

Subacute Sclerosing Panencephalitis or SSPE is one of the most serious complications of measles resulting from viral infection of the central nervous system; SSPE is rare (1 in 10,000-25,000 measles infections) but is almost always fatal. Following infection at a particularly young age and on average 8 years following acute infection, a progressive deterioration of neurological function presents : loss of attention span, uncontrolled movements, behavioural changes, cognitive impairment and in all cases vegetative state is entered and death occurs.

It is caused by persistent measles infection i.e one that the isn't removed when your immune system kicks in, which spreads throughout the  cells found within the brain causing cell death and inflammation. Strangely, no infectious virus can be recovered from infected brains and when this was investigated further they found that many mutations occurred throughout the genome rendering many of the genes nonfunctional. Although the major replicative functions (replication and gene expression) were left intact, the genes required for normal particles formation were those mutated suggesting that the virus may exploit the unique cellular environment in the CNS to spread, replicate and survive.

[caption id="" align="aligncenter" width="402" caption="Green Fluorescent Protein expressing measles virus infection of neuronal cell"][/caption]

As I mentioned previously, due to increased transmission of virus, poverty and poor nutrition, measles infection is extremely serious in developing countries and it is no surprise that SSPE occurs here in higher numbers. In Papua New Guinea there exists a very high incidence of SSPE, THE highest incidence - roughly 3 - 20 times as many cases are reported (98 per million people versus 5 per million people). Manning et al (2011) have attempted to further characterise SSPE behaviour in this country between 1997 and 2008 and highlights the significant burden that measles is in many developing countries. They measured SSPE incidence, measles infection rates and time of birth of each patient presenting with SSPE finding a direct correlation between time of birth, measles epidemics and presenting with SSPE. The group emphasises the requirement

Why is SSPE incidence so high here and what can we do about it? SSPE rates are linked to measles infections in a population and hence have been significantly reduced following measles vaccination campaigns. Sadly, only half of children in Papua New Guinea receive two measles vaccines prior to 1st birthday - not enough to sufficiently protect an individual nor a population from measles infection and hence SSPE; there is insufficiently low-level of herd immunity in regions such as papua New Guinea. The level of vaccine effectiveness of measles vaccine in this region is also particularly low - possibly reflecting damage to the vaccine from cold-chain disruption (in tropical climates it is difficult to keep vaccines refrigerated), population genetic effects or persistence of low-level non-neutralising maternal antibody.

We can no longer afford to ignore the importance of measles in developing countries like Papua New Guinea and we must stress the need for adequate vaccine effectiveness and coverage in already susceptible human populations. Studies like these with SSPE emphasise the real-world need for the investigation of the molecular mechanisms of measles virus persistence and we should look forward to a time when we can adequatly treat measles CNS complications - or maybe with better vaccination coverage we may not have to worry about this.

Manning, L., Laman, M., Edoni, H., Mueller, I., Karunajeewa, H., Smith, D., Hwaiwhanje, I., Siba, P., & Davis, T. (2011). Subacute Sclerosing Panencephalitis in Papua New Guinean Children: The Cost of Continuing Inadequate Measles Vaccine Coverage PLoS Neglected Tropical Diseases, 5 (1) DOI: 10.1371/journal.pntd.0000932

Rima, B., & Duprex, W. (2006). Morbilliviruses and human disease The Journal of Pathology, 208 (2), 199-214 DOI: 10.1002/path.1873

A mothers love declines - a measles vaccine problem?

Worldwide, measles virus infection accounts for around 200,000 deaths annually; the importance of which is emphasized given the availability of a highly effective vaccine. Vaccine effectiveness, however, is a complex matter and is subject to many problems – a major one being transfer of maternal antibodies to children during early life, a form of natural passive immunity. Although these antibodies are there for a reason and do protect offspring from infections in early life, bridging the gap until they can synthesize their own antibodies, they have been shown to inhibit the activity to certain vaccines – measles vaccine is an example (see figure below).

[caption id="" align="aligncenter" width="381" caption="Antibody concentrations in child following birth showing decline in attenuation (infection attenuation)"][/caption]

During early childhood, maternal antibody concentrations begin to wane and eventually reach such a level as to offer little protection from microbial challenge. These antibodies however are able to dampen the ability of a child to develop protective immunity following vaccination; it is this ‘window of opportunity’ that is responsible for a great number of measles virus infections and fatalities every year. The development of an effective vaccination strategy to get around this blocking effect would therefore be of great medical interest.

Recently, Kim et al (2010) publish their investigations into understanding how and why measles virus infection, in the presence of specific antibody results in the inhibition of a protective response following vaccination. Prior to this study it was unknown whether in this situation, MeV-specific B cells were being generated at all or whether they simply failed to secrete neutralizing antibody. The group used a rat model of MeV infection and simulated maternal antibody effects by passively transferring MeV specific antibodies and measuring the immunological outcomes. They demonstrated that there is a specific failure of B cells to secrete protective antibody in the presence of transferred antibody.

B cells will only secrete antibody when 3 signals are triggered:

1.     B-cell receptor/antigen interactions

2.     B – cell/ T-cell interactions

3.     Action of soluble mediators (for example: cytokines like interferon)

Kim et al hypothesized that in this model, where both signals 1 and 2 were active, inhibition of antibody secretion may be accounted for by the interference with certain soluble mediators. This idea was attractive providing the great deal of evidence showing MeV obstruction of interferon production - a pathway that normally results in the robust development of innate and adaptive immune responses. This results in two major problems involving antibody-specific inhibition of protective immune responses (maternal antibody) combined with MeV’s natural ability to inhibit the development of immunity; cases which are shared during the ‘window of opportunity’.

To this effect, the group developed a novel vaccine vector to circumvent wild-type measles interferon inhibition. Using reverse-genetics technology, they incorporated a MeV antigen gene, the haemagglutinin (HN) glycoprotein into the Newcastle Disease virus (NDV) genome as an extra gene, generating NDV-HN. NDV is an avian virus that induces high concentrations of IFNs upon infection allowing for the possibility of an effective measles vaccine in the presence of measles antibody.

The investigation confirmed the group’s predictions in that NDV-HN induced much higher levels of IFN in rat tissues when compared to MeV and that this led to development of MeV-specific neutralizing antibodies in the presence of transferred antibody. This work further verified that role that the restoration signal 3, in the form of alpha-IFN allows for B-cell secretion of antibody in in vivo and in vitro systems.

Given how medically important vaccination has been in protecting populations from often fatal and serious infectious disease and the troubles that arise when maternal antibody concentrations drop, any work developing vaccine technology to avoid these difficulties should be welcomed. Kim et al. confirmed the basis for the immunological blocks in generating MeV antibodies and began the development of a novel vector system to rationally provide protection. The results in this rat model, although not specifically applicable to the human situation, are promising in that it provides a logical framework to advance vaccine technology and prevent thousands of childhood deaths worldwide.

Kim D, Martinez-Sobrido L, Choi C, Petroff N, García-Sastre A, Niewiesk S, Carsillo T. (2011) Induction of Type I Interferon Secretion through Recombinant Newcastle Disease Virus Expressing Measles Virus Hemagglutinin Stimulates Antibody Secretion in the Presence of Maternal Antibodies. J Virol. 2011 Jan;85(1):200-7. PMID: 20962092