Towards a malaria vaccine: Indian researchers are making progress

19 Nov 2008

Tatum Anderson

Source: TropIKA

Comments

Dyann Wirth
Dr Chetan Chitnis

The path towards one of the most promising vaccines to emerge from India in recent years was set in motion thousands of miles away in 1991.

Dr Chetan Chitnis, a postdoctorate scholar from India joined a research group at the National Institutes of Health (NIH) in the US, led by Louis H. Miller, an expert in the pathogenesis of malaria and chief of the Malaria Cell Biology Section at the Laboratory of Malaria and Vector Research.

'I was looking for post-doc work and thought I should work on a tropical disease because I had a better chance of finding work back in India,’ says Chitnis. 'That's how I got into infectious diseases relevant to developing countries and found myself at the NIH working on malaria in a really excellent lab.'

That laboratory had already achieved a major breakthrough in research on the malaria parasite called Plasmodium vivax which is responsible for over two thirds of cases in India. Less deadly than Plasmodium falciparium, P. vivax causes serious recurring malaria throughout Asia, Latin America, and Africa. Indeed a person living in a moderately P. vivax-endemic area can experience as many as 30 episodes of malaria in a lifetime.

Miller's group had discovered that a protein, called the Duffy blood group antigen, is receptor for P. vivax. In other words, that the malaria parasite latches onto the antigen, which appears on the surface of red blood cells, and uses this protein to prize open the blood cells, invade them and reproduce.

It was a hugely important discovery says Chitnis. 'P. vivax is absolutely dependent on the Duffy antigen for invasion if you don’t have Duffy you don’t get invasion. There is only one pathway. It's absolutely essential,' he says.

Miller's group had taken a step further and discovered that a very particular part of the parasite was responsible for binding to the red blood cell. It was called the Duffy binding protein, because it attaches to the Duffy antigen on the blood cell.

By the time Chitnis joined the team, it had worked out how to clone the gene that expresses the parasite's Duffy binding protein. His job was to discover exactly what part of a very large protein could bind with the red blood cell.

'The protein is about 150 kilo-Daltons [about 150,000 times heavier than a hydrogen atom] and we narrowed down the binding region to about 40 kilo-Daltons,' he says. 'The protein is divided into six different regions, and region two we found was the binding domain.'

That information was key to the creation of a potential vaccine, and determined the focus of his work when Chitnis returned home to India. He has since spent a decade seeking to perfect the first malaria vaccine to target this Duffy binding protein.

ICGEB

Chetan Chitnis is based at the International Centre for Genetic Engineering and Biotechnology (ICGEB), an international research organisation that was set up in the late eighties to further agriculture and health-based biotechnology in developing world. With three headquarters - in Italy, India, and more recently Cape Town - this major collaboration brings together scientists from 63 countries predominately from the developing world.

At the labs in New Delhi, Chitnis is one of a large group, headed by Professor Virander Chauhan, that is dedicated to investigating malaria. Other groups at ICGEB are studying HIV, hepatitis and influenza viruses. The ICGEB immunology group is now working on tuberculosis.

Chitnis' work at ICGEB has focused on reproducing Region II of the parasite protein that binds to red-blood cells using biotechnology. The idea is that if scientists can create a vaccine made from such copies of this fragment of the protein and inject them into humans, the body will be stimulated to create antibodies. The ultimate aim is to ensure that if an immunised person becomes infected with malaria parasites, these antibodies will bind this Duffy binding protein on the parasite and prevent it from invading red blood cells and reproducing.

The problem is that reproducing the parasite sections is very difficult. That is because its structure extremely complex and made up of hundreds of amino acids, with unique physical chemical properties. Some amino acids are attracted to water, for instance, while others repel it. Some are positively charged and others are negatively-charged. These properties are critical to the protein’s structure because together they influence the way the protein folds, and how it interacts with blood cells. The key for Chitnis’ team is to ensure the copies act in exactly the same way as the parasite would, thereby stimulating an immune response.

Therefore, Chitnis' team has been devising ways of reproducing the fragments of the protein responsible for binding re-creating that fragment’s structure and then checking that it binds correctly. ‘We found a 350-amino-acid region which you can produce on its own and it retains the binding properties to the red cells,' he said.

While it is possible to grow the protein using the gene combined with the bacteria E. coli, it is no trivial process to re-create the intricate protein fragment structure, says Chitnis.

'We put the gene into E. coli and made lots of fragments but it doesn't fold out, so we have created the methods to re-fold the protein. It took a few years to work that out,' he says.

The team has now solved this problem, devising a method to create the fragment. It has been working with an industrial partner, Indian biotech firm Bharat Biotech, to develop industrial-grade fragments on a larger scale.

But recreating the fragment has not been the team's only task. It has worked hard to build up the rationale for using this particular fragment as a vaccine main ingredient.

That has involved establishing whether fragments do in fact stimulate antibodies that protect against P. vivax invasion with collaborators in Papua New Guinea.

The researchers have also checked whether the fragment will work against the different strains of P. vivax. Genetic diversity results in slight variations in the 350 amino acids of the fragments from parasite to parasite. In theory some variations might help a parasite to evade antibody responses.

As a result, the group worked with ICGEB's X-ray crystallographers to determine the precise 3D structure of the region in order to narrow down precisely which amino acids are involved in binding.

That analysis threw up a rather startling and fortunate discovery, says Chitnis. 'We found that actually, although there are differences in the amino acid sequences and the specific units that make up this domain, the residues involved in binding are conserved and are always the same,' he says. 'That came as quite a surprise and that's actually very good news because it means this vaccine should work against all strains.'

Now, the priority is to secure a good adjuvant, a chemical that stimulates the immune system to find and react to the vaccine.

That has not been easy either. The most commonly used - and cheapest - off-patent adjuvant is alum, which is present in most vaccines. However alum is not ideal for a malaria vaccine says Chitnis. So, the researchers have been trying to negotiate access to on-patent adjuvants on reasonable terms. Some negotiations have been more fruitful than others, as different pharmaceutical corporations appear to require different incentives to share intellectual property with public health projects such as ICGEB. Chitnis is, however, confident that an adjuvant will be found in due course.

Can a vaccine be effective against vivax and falciparum?

Focusing on P. vivax is unusual; most vaccine scientists across the world are frantically trying to find candidates to prevent the deadlier P. falciparum.

Indeed there are there are about some 40 P. falciparum candidate malaria vaccines or vaccine components in the pipeline and only a few for P. vivax. ICGEB's is the only one vaccine for P. vivax.

But vaccines targeting both types of parasite are of immense importance in India, says Chitnis. Here many sections of the population - over 80% in some areas, suffer from P. vivax. (In areas of Africa there are places where 95% of people will not be infected with P. vivax for genetic reasons, he says)

'In India, we need vaccines for both because, if you make a vaccine for one and people still get infected, the vaccine would be perceived as being a failure,' he says.

A focus on P. vivax is even more critical now that there is an international emphasis on eradicating P. vivax altogether after global players including the Roll Back Malaria partnership created an eradication roadmap in September. 'There is equal emphasis on both species. Now there is awareness that we need to deal with both and so hopefully there will be more support for P. vivax.'

Indeed in a statement from the recent eradication roadmap states: 'A vaccine for P. vivax is increasing in development priority. Vaccines which target P. vivax may in fact prove to be necessary to achieve elimination and eradication.'

P. vivax is not the only focus of the ICGEB malaria team's work, however. They are looking at potential antigens for P. falciparum, the most deadly species of malaria parasite.

They've used the same principles that lead to the creation of the P. vivax candidate. In other words, finding one parasite protein that binds to a red blood cell and then re-creating the fragment of the parasite protein that binds. But P. falciparum is far more complex than P. vivax, because there are hundreds of different mechanisms believed to be used by the parasites to invade the body.

As a result, the researchers are combining two different fragments. But this approach is unusual, because most groups are testing one antigen at a time. But testing samples with mixed antigens now is critical, says Chitnis. 'There are questions when you combine to antigens - does the antibody response get skewed against one or do you get a balanced response to both?' he says.

It has certainly been a learning curve to convert the product of years of academic research into something that is ready for testing in humans. 'Getting the vaccine made and tested is quite a challenge, since we're trying to do it in an academic setting. I know a lot more today than I did five or ten years ago but still there's a long way to go to be able to do a field trial,' says Chitnis. 'The good thing is that now there is funding for malaria vaccine development and more people doing this at a global level, so you can establish collaborations to get things done.'

Key Questions

Briefly, what are the priority concerns of your organisation? And, more precisely, what goals have you set?

To develop a vaccine for P. vivax vaccine PvRII based on Duffy binding protein, another P. vivax vaccine based on MSP-1 and a P. falciparum vaccine JAIVAC-1, which is a combination of PfF2 (EBA175) and PfMSP1(19). On track for Phase I clinical trials next year.
What is it about your organisation's approach to these issues that distinguishes you from others in this field?

We put equal emphasis on our vaccine programme for P. vivax and P. falciparum, because in a country like India, we have a very complex large geographical area and need to cover both.
What are the main challenges outstanding?

To identify an adjuvant and start Phase 1 Clinical trials. The next challenge is to develop a pipeline of new candidates and improve the understanding of the basic biology and the components of the immune response that provide protection. There is a lot of basic research that needs to keep happening which will keep that pipeline of vaccines full.
Which other organisations will you be working with most closely?

ICGEB has been working closely with EMVI, Bharti Biotech and numerous national and international partners, including the Government of India's Department of Biotechnology, the World Health Organization's Tropical Disease Research Division, the Indo-US Vaccine Action Program, an alliance between the Department of Biotechnology and the US National Institutes of Health and the PATH (Program for Appropriate Technology in Health) Malaria Vaccine Initiative (MVI). A new grant is in process, so partners are subject to change.

Further reading

Miller LH, Mason SJ, Clyde DF, McGinniss MH (1976). The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med; 295(6):302-304. http://www.ncbi.nlm.nih.gov/pubmed/778616
Miller LH, Mason SJ, Dvorak JA, McGinniss MH, Rothman IK (1975). Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants. Science. 189(4202):561-563. http://www.ncbi.nlm.nih.gov/pubmed/1145213
Horuk R, Chitnis CE, Darbonne WC, Colby TJ, Rybicki A, Hadley TJ, Miller LH (1993). A receptor for the malarial parasite Plasmodium vivax: the erythrocyte chemokine receptor. Science; 261(5125):1182-1184. http://www.ncbi.nlm.nih.gov/pubmed/7689250
Sim BK, Chitnis CE, Wasniowska K, Hadley TJ, Miller LH (1994). Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum. Science; 264(5167):1941-1944. http://www.ncbi.nlm.nih.gov/pubmed/8009226