Feature: Fever pitch

This issue appeared in the November/December 2011 edition of Australian Life Scientist. To subscribe, click here.

First isolated in Bankgok in 1958, the chikungunya virus causes endemic disease in urban areas of Africa and epidemic infections in parts of Central and Southeast Asia, India, Papua New Guinea and Europe. Although usually associated with low mortality, a 2005 outbreak on Reunion Island, near Madagascar, killed more than 200 people.

Unlike Ross River virus, named for the eponymous river in North Queensland, the chikungunya virus is not yet found in Australia, although some experts believe it is only a matter of time, particularly given recent confirmation that native mosquitoes can be infected when exposed to the virus.

At the Australasian Society for Immunology conference in December, Professor Suresh Mahalingam will talk about his work on the mosquito-borne alphaviruses, specifically chikungunya and Ross River virus, which are known to cause significant inflammatory pathologies ranging from arthritis to encephalitis.

Mahalingam and his team are aiming to understand the mechanisms by which viruses like chikungunya and Ross River virus cause disease, which remain poorly understood, in the hope of improving treatment and prevention of the diseases.

Ross River virus and chikungunya virus cause a clinically identical musculoskeletal disease characterised by fever, myalgia (muscle pain), arthritis, crippling arthralgia (joint pain), rash and severe lethargy.

The infection often leads to chronic joint inflammation and debilitating pain, commonly affecting ankles, hips, wrists and knees, and sometimes lasting for months to years. The symptoms are very similar to those in sufferers of rheumatoid arthritis.

“We would like to decipher the mechanisms of these diseases and from that, try to identify better drug targets that will be useful in alleviating the disease,” says Mahalingam. “Currently, there is no licensed vaccine available for preventing chikungunya or Ross River viral disease, and current treatments are symptomatic, using broad-spectrum anti-inflammatory drugs.

“The problem with these non-specific agents is that they might reduce the inflammation but, at the same time, may also suppress some of the proteins and mechanisms that are important for the innate immune system to reduce infection and eventually clear the virus – even potentially exacerbating the infection.

“So, we have developed and used several animal and human cell models to identify quite a few really cool new pathways that we can target to reduce the disease symptoms without affecting the ability of the host to clear the infection.”

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Old protein, new function

At ASI, Mahalingam will also present some new and exciting results that are about to be published. “Using our models of chikungunya virus infection, we identified a pathway and a specific protein in that pathway whose function in this context has never been reported before.”

They then blocked the function of this protein and, hey presto, successfully reduced the damaging pathology caused by the infection. It also seems that the mechanism involved is not necessarily unique to alphaviruses, and it may potentially be a contributing factor for Dengue and other important human viral infections.

Mahalingam’s new protein find belongs to the huge and complex complement system, which was first identified three decades ago. The complement system mediates the innate immune response in humans by helping antibodies and phagocytic cells to clear pathogens. Over 25 proteins and protein fragments make up the complement system, with many more in the complement-activated signaling pathways.

“From the literature, we know that this protein is polymorphic in terms of its expression. In other words, different individuals express this protein at different levels, and 10 per cent of people do not express it at all,” says Mahalingam.

And because this protein has a role in innate immunity, those people who make none of the protein or only a little are immunocompromised, at least to some extent, and therefore easy prey to the establishment of an infection if challenged.”

However, what Mahalingam’ team found with their models was that in the absence of this protein, the chikungunya viral immunity was not actually compromised. “In fact, there was no difference detected in virus replication in hosts with and without expression of this protein, or between those mice showing low and high expression.

“What was even more interesting was that, in the absence of the protein, the infection-induced disease was basically lost. No musculoskeletal tissue breakdown or other disease pathology was detected in mice not expressing the protein, and this was despite the virus being able to replicate to high levels equal to the wild-type mice.”

The results from Mahalingam’s work therefore implicated this protein, which is important for maintaining a generally immunocompetent state, in actually mediating the tissue damage caused by a chikungunya virus infection.

“In the absence of this protein, the inflammation is still there, with all the usual suspects in terms of pathogen-detecting and -killing cells and mediators, but for some reason the tissue remains healthy and shows no damage, and the mouse overall seems quite healthy.”

In very recent work, Mahalingam now has evidence from human data that correlates to the mouse story concerning this protein. “We believe that this protein mediator from the complement pathway somehow plays a role in driving the tissue damage associated with these arthritogenic viral infections, probably by mediating events upstream of the tissue damage process. This is a completely new finding.”

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To market

To go down the commercial path with this finding, the team is now embarking on proof-of-principle studies with a humanised monoclonal antibody that they have developed against their novel protein target. If that is successful, they will file a patent and go from there.

“We believe that this finding could be quite significant because, if this protein is acting this way in inflammation cause by a viral infection, it could also be applicable to other viral and even bacterial infectious disease in different parts of the body,” says Mahalingham.

“By blocking the tissue pathology you could limit the severity and illness from the infection, because it is the tissue damage and not the actual inflammation that kills people and causes disease. Then the body can work on clearing the infection itself. So, that is the goal.

“As part of this work, we are also looking at existing drugs on the market that may affect the viral-mediated pathways and proteins we identify as candidate targets.” Mahalingham gives recent examples of how this ‘recycling’ approach has already proved quite successful.

The first involved targeting a protein belonging to the monocyte chemotactic protein (MCP) family. MCP-1 is responsible for recruiting macrophage migration into the virally infected tissue, and it is these immune cells that kick off the tissue-damaging inflammation and associated symptoms in the infected individual.

“So we did a screen for agents that target MCP-1 and found this company in Italy (Angelini Pharmaceuticals) that produced a small molecule anti-inflammatory drug for treating nephritis – called bindarit. This drug actually works by wiping out all the MCP proteins and, better still, it can be given safely to humans without causing toxicity.”

Mahalingam then wrote to the company and obtained some bindarit to test in their mouse model of chikungunya viral disease. To their delight, the researchers found that infected mice given this drug showed less musculoskeletal inflammation than their untreated counterparts. This was the first confirmation of MCP-1’s key role in chikungunya virus-induced arthritis and highlighted the therapeutic potential of MCP inhibitors.

“We were happy because this approach expedited the drug development process for such an agent by at least four to five years due to the fact that it is already on the market for human use. And the company was happy because they can add another indication to their list for bindarit.” Clinical trials to test the effectiveness of the drug in relieving chikungunya viral disease are now being conducted in two hospitals in western India.

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The second and more recent example of Mahalingam’s drug-detective approach is set to make an important contribution to clinical treatment. “We also started looking at a drug currently on the market to treat rheumatoid arthritis.

“Called etanercept, this drug neutralises the key pro-inflammatory mediator, tumour necrosis factor (TNF). We speculated that this anti-TNF drug could contribute to the viral arthritis pathology in the same way for inflammatory diseases like rheumatoid arthritis.”

The team ran a study to evaluate etanercept for treating virally induced-arthritis in their mouse model of rheumatic disease. “We got some of this drug and tried it, but instead of making them better, it killed our mice within three to four days.”

It seemed from their surprising results that anti-TNF treatment could actually be detrimental to patients with viral arthritis, although it was consistent with numerous reports of rheumatoid arthritis patients suffering from severe viral infections.

According to Mahalingam, the message to clinicians is simple: in places where viral infections like chikungunya or Ross River are endemic and people have rheumatoid arthritis or similar, do not use anti-TNF treatments.

This issue appeared in the November/December 2011 edition of Australian Life Scientist. To subscribe, click here.