T. V. Jayan*

 

In what could be described as a significant achievement in the fight against anthrax, a potent biological weapon, Indian scientists have bio-engineered a new vaccine which is less toxic and more effective than those currently available in the market.

The candidate vaccine, developed by a team of researchers, led by Prof. Rakesh Bhatnagar, Chairperson of the Centre for Biotechnology at the Jawaharlal Nehru University (JNU) in New Delhi, has already been transferred to pharmaceutical firm Panacea Biotech Ltd in the first week of November, for commercial production.

The JNU teams feat was widely acclaimed as the announcement came at a time the world was in the grip of an anthrax scare after five suspected anthrax related deaths in the United States in October and November. A numerous government and media offices in the U.S. were reportedly flooded with mails containing anthrax spores, in the aftermath of the September 11, 2001 attacks in News York and Washington.

What distinguishes the Indian candidate anthrax vaccine from those currently in use is its relatively less or no toxicity. The anthrax vaccines available today, when used for immunization, require a number of booster doses and have several undesirable side-effects.

Anthrax is a disease of grazing animals. The rod-shaped bacteria, B. anthracis, spend most of their time as spores in the soil, encased in a tough shell and practically lifeless. But when an animal swallows or inhales these spores, they come to life, and exude a protein which allows them to get into the white blood cells called macrophages. Though one of the major functions of macrophages is to destroy bacteria, fast-multiplying anthrax bacilli outmaneouvre macrophages, and eventually leading to the death of the animal. Human beings who come in contact with animals that die of anthrax have a greater risk of contracting the disease.

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Prof. Rakesh Bhatnagar

Dr Yogendra Singh

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Even though a 19th century scientist Robert Koch postulated the existence of toxins of B. anthracis, it wasnt really known what actually enables the bacteria to cause harm to animals and human beings till H Smith and J Keppie unravelled the molecular mechanism behind the infection in 1954. They discovered that the anthrax toxin complex consists of three proteins: a cell-receptor binding protein known as protective antigen; and two enzymes, lethal factor and edema factor. Protective antigen central component of anthrax toxin makes it possible for the other two components to enter into the host cell. Subsequently, it was found that these proteins are not toxic all by themselves, but when lethal factor and edema factor are combined with protective antigen, they form potent toxin.

This knowledge helped scientists to develop many vaccines against anthrax in the past, one of the more evolved so far being a cell-free filtrate vaccine, which was used by the US to immunize its armed personnel. A cell-free filtrate vaccine means that the vaccine contains no dead or live bacteria unlike live attenuated vaccines.

The Indian candidate vaccine, however, is claimed to be superior to the one being used by the U.S. Army. The Indian scientists reportedly managed to nullify several side-effects associated with anthrax vaccines by genetically modifying the proteins protective antigen, lethal factor and edema factor. The scientists not only rendered these proteins to be used in the vaccine preparation harmless, but also successfully over-expressed them in safer hosts. They also manufactured a few grams of anthrax proteins in an experimental bioreactor in the laboratory, before passing the technology over for commercial production to Panacea Biotech Ltd, through Biotech Consortium Limited, an organization set up to commercialize the technologies developed through the Department of Biotechnology (DBT) funding. Few grams of purified anthrax protein are equivalent to million doses of currently available vaccine, according to Prof. Bhatnagar. The effort to develop an Indian anthrax vaccine had actually begun in 1995 with the DBT sanctioning a research grant to Prof. Bhatnagar and Dr. Yogendra Singh of the Centre for Biochemical Technology, a research laboratory under the aegis of the Council of Scientific and Industrial Research (CSIR).

Dr. Singh, who associated with the project in the early stages of the project and helped develop harmless mutants of anthrax proteins, has been working on anthrax for 14 years in various Indian and U.S. laboratories. Recently, he independently developed a therapeutic agent, which is capable of neutralizing the effect of the anthrax toxin in post-antibiotic treatment. This molecule, for which he recently filed a U.S. patent, is a potential solution to one major hurdle in the treatment of anthrax ineffectiveness of drugs after setting in of symptoms.

Dream 2047 spoke to Prof. Rakesh Bhatnagar to know more about candidate anthrax vaccine developed by his team. Following are the excerpts of the interview with Prof. Bhatnagar:

Dream 2047: Anthrax, a disease originally thought to affect animals, suddenly assumed significance with reports of several cases of fatalities in the U.S soon after the September 11 attacks. What is the treatment currently available for anthrax? How significant is the role of vaccination in managing anthrax?

Prof. Bhatnagar: Antibiotics such as ciprofloxacin and penicillin are found to be effective in treating anthrax. But what makes them unpopular is the long duration of the treatment it takes about two months to cure the disease. Besides, the treatment is not quite effective once the symptoms of the disease have set in. Hence, like most other diseases, in the case of anthrax too, prevention is better than cure.

Dream 2047: But there is another school of thought which says that vaccination may not be an absolute necessity in the case of anthrax as the disease is seldom widespread?

Prof. Bhatnagar: This is not true. Take the case of a poor farmer. Suppose one of his cattle infected with anthrax dies. And he peels off its skin to earn a little extra money. There is a probability that he might contract the disease in the process. If he consults a doctor complaining uneasiness, the chances are high that the doctor might prescribe parasitamol or some such drug to him. This is because anthrax has symptoms very similar to that of common cold breathlessness, pain and fever. In such a scenario, it will be very difficult to save him, considering that inhalation anthrax one of the most virulent forms has a fatality rate of about 95 per cent.

Dream 2047: Anthrax vaccines have been in the market for quite some time. It is said that Louis Pasteur was the first scientist to develop a vaccine against anthrax about 120 years ago in 1881. How is the vaccine developed by your team at the Jawaharlal Nehru University is different from those currently available in the market?

Prof. Bhatnagar: What Pasteur developed in the 19th century was a live attenuated vaccine against anthrax. This means the bacterium itself was injected to develop immunity. In the early 50s, scientists identified three molecules responsible for the virulence. They are protective antigen, lethal factor and edema factor. These proteins all by themselves are not toxic. But when protective antigen combines with lethal factor, lethal toxin is generated. Similarly, mixing edema factor with protective antigen creates an edema toxin, which causes swelling and redness of the skin. The preparations (other anthrax vaccines) currently used contain protective antigen, which is the principal immunogen, and traces of lethal factor and edema factors. These preparations have several undesirable side effects due to contamination of these proteins.

On the contrary, we introduced mutations into all three proteins of the anthrax toxin complex, rendering them non-toxic and free from side effects. In order to create an improved vaccine, we have introduced the genes for mutated proteins into relatively safe host organisms.

Dream 2047: At what stage is the development of new vaccine? When do you think the vaccine will be available for use?

Prof. Bhatnagar: We have intensively studied the candidate vaccine at the cellular level. The lab scale technology has now been transferred to Panacea Biotech Ltd through Biotech Consortium India Ltd. This pharmaceutical company will conduct animal toxicity and clinical trials. They have collected the clones and the molecules from the lab and will produce the vaccine in larger amounts. Then they will have to do animal toxicity studies and subsequently the efficacy studies. Once they are ready with the data from the animal trials, they would approach the Drug Controller of India (DCI) for permission to carry out clinical trials on human beings. We hope that the Indian anthrax vaccine will be in the market in nine months time. All that but will depend on successful completion of various trials on animals and human volunteers and the subsequent clearance from the DCI.

Dream 2047: How expensive would the vaccine be?

Prof. Bhatnagar: The new anthrax vaccine is certainly going to be much cheaper than those currently available in the market. Even the five-litre table-top bioreactor, available at our laboratory, can produce 5 grams of protective antigen per litre per day. This is enough to manufacture one million doses. The company is setting up a much larger capacity. The economy of scale will further drive down the price.

Dream 2047: We are curious to know what made you you as well as Dr. Yogendra Singh to work on anthrax vaccine.

Prof. Bhatnagar: Both of us commenced our work on anthrax when we were in a U.S. laboratory several years ago. I was actually working on the molecular mode of action of the anthrax toxin. What prompted us to work on anthrax even after returning to India was outbreak of an anthrax epidemic in West Bengal in 1994. Subsequently, we submitted a proposal to develop an anthrax vaccine to the Department of Biotechnology and this was approved in 1995.

This July, our team at the Centre for Biotechnology submitted a paper on the vaccine to the American Society for Cell Biology and it was accepted for a conference in Washington on December 8-12, 2001. The ASCB, in the aftermath of anthrax scare in the U.S., released the findings to the public through its website www.ascb.org in October itself, and requested me to address a press conference during the meeting.

Dream 2047: What do you feel about this achievement?

Prof. Bhatnagar: First of all we are very happy that we could achieve what we had set out for. Moreover, it is often said that quality scientific work is difficult in India. The appreciation our work received from the peers both in India and abroad shows that quality work can be done in India, provided there is a conducive environment. We are certain that this could not have been achieved without unwavering support that we received from the Department of Biotechnology, an excellent environment that exists in Jawaharlal Nehru University and above all the consistent effort of students who work in our laboratory.

* T.V. Jayan has recently joined Vigyan Prasar as a Fellow.