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New vaccine against rabies

The Butantan Institute creates a safer, cheaper and more efficient alternative for use in human beings

Secure, efficient and cheap, a new vaccine against human rabies should be launched by the Butantan Institute over the next few months. Developed in its entirety at the Paulista institution, the product, which has already been tested with success on mice and monkeys, has passed through its final test: it was injected into more than 200 human beings in a study carried out by the Pasteur Institute of Sao Paulo and the results, as yet preliminary, were good. There were no significant side reactions of an allergic or nervous type, and in  the majority of patients the vaccine stimulated levels of antibodies thirty (30) times higher than that considered sufficient by the World Health Organization (WHO) in order to neutralize the action of the rabies virus. The quantity of residual cells found in the immunizer doses was very low, close to five times less than that recommended. Rabies is a fatal disease to man if untreated shortly after the infection, via the pathogen, has occurred.

The authorization for the sale of the vaccine, whose intellectual property rights have been protected by patents since the year 2000, are going to be requested this month from the National Agency for Sanitary Vigilance (Anvisa). Over the next few months, all of the necessary documentation for the approval of the medicine will be sent on to Brasilia. “Until the end of the year, or the start of 2006, we’re going to begin the production of the vaccine on a commercial scale”, affirmed Dr. Neuza Maria Frazatti Gallina, head of the rabies sector at Butantan, responsible for the product’s development. Initially the goal will be to manufacture around 3 million doses of the vaccine annually, sufficient to attend to national demand. In a second phase, the production volume will be elevated, looking towards exportation. The estimated cost of each dose is US$ 5, two US dollars less than the price paid by the federal government for the rabies vaccine currently used in the country, imported from France and afterwards tested and labeled by the Butantan Institute.

The product’s quality is derived from the domination of an unprecedented form of cultivating the rabies virus, the infectious agent belonging to the Lyssavirus genre. The raw material for the confection of the vaccine, where it is present in an inactive form, is the virus grown in a substrate composed of the so called Vero cells, removed from the kidneys of the Vervet monkey [African green monkey] (Cercopithecus aethiops). One is dealing with a very stable type of biological material, without the risk of bringing about health problems in humans, which can be obtained from an international bank of Vero cells. Because of these characteristics, and also because of the possibility of growing them in high concentrations in the interior of huge bio-reactors, these cells come highly recommended by the WHO in the production of vaccines. They are capable of multiplying themselves in a culture environment for cell growth that dispenses with the need for serums of bovine or human origin. Indeed, there are fewer traces of animal DNA in the final vaccine compound.

Through this process, the chance of manufacturing a contaminated vaccine, for example, with a degenerate form of prion, a bovine protein that causes mad cow’s disease, is nil. “We’re the first in the world to be making a vaccine against rabies from Vero cells within a medium free of serum, a technique that’s normally much more expensive”, advised Dr. Neuza, who, in her work was able to count upon financial support from FAPESP, the Butantan Foundation and the National Council for Research and Technological Development (CNPq). “But with our method the production is five times more efficient than the other methods, and what was once expensive becomes cheap.” Made abroad, the human rabies vaccine today available in Brazil also makes use of Vero cells in its production method, but is obtained starting from the culture of the rabies virus in a medium that still needs animal serum.

The adoption of an imported vaccine was the stop-gap solution to remedy the situation that the country found itself in at the end of the decade of the 1990s. At that time there was a national version of rabies vaccine available, made by the Butantan Institute and the Technology Institute of Parana (Tecpar), the only thing was that is was derived from a production technology that was very old. It was a vaccine whose manufacturing process made use of the brains of recently born mice. Around 2% of its final content was brain tissue from rodents. “The old formulation of the national vaccine was good and conferred immunity, but the risk of adverse side effects could not be ruled out”, commented Dr. Neuza.

At the end of the decade of the 1990s, one person in the country after having taken a dose of the old vaccine showed serious neurological reactions and died. During the year 2000, the State of Sao Paulo banned the manufacture of the product that had come from the nerve tissue of mice. Two years later, the federal government did the same thing. With nobody in Brazil then dominating the technology for making purer vaccines against rabies, the solution was to bring a safer medicine in from abroad. The new Butantan Institute vaccine, even safer than that currently imported, should put an end to the temporary need for importing the immunizer, guaranteeing new technological independence to the country in this area. Into the bargain, the studies of more than a decade that have led to the new anti-rabies vaccine for human use will also allow for the development of a more modern lineage of immunizers against rabies that attack dogs, cats and cattle.

The Tecpar Institute also pretends to develop a human anti-rabies vaccine starting from a culture medium of Vero cells, but their researchers are at an earlier phase than that of the Butantan Institute. “As yet we can’t manage to produce the vaccine in the medium free of serum on an industrial scale”, advised the biochemist Renato Rau, the production director at the Tecpar Institute, which recently has associated itself to an Argentinean company in the hope of dominating the process. The control of the technology of culture via a cellular medium free of serum will be useful to the Butantan Institute in the creation of other pharmaceutical products, such as a national version of the vaccine against the rotavirus, which causes common diarrhea in children. “This is our next challenge”, advised Dr. Neuza.

The production process of an industrial batch with 120,000 doses of the Butantan Institute anti-rabies vaccine is rapid: it takes only nine days. In a 30 liter capacity bioreactor, which continuously stirs the content at a speed of 60 rotations per minute, the rabies virus is reproduced when in contact with the Vero cultured cells in a medium free of serum under controlled conditions for a series of parameters such as temperature, quantity of oxygen and acidity (pH). Periodically samples are collected: a little of the solution rich in the rabies virus in the bioreactor is removed, to which more of the culture medium is added. Such a procedure is repeated six times until the end of the manufacturing process of a vaccine batch. Finally, the viral suspension is concentrated, purified and the pathogen within it is made inactive. After final preparation the new vaccine can be stocked for 14 months at a temperature between 2ºC and 8ºC. More details concerning the new immunizer can be obtained in an article published by the Butantan Institute researchers last December in the scientific magazine entitled, Vaccine.

Rabies is a zoonosis, an illness transmitted by animals to man (and to other animals). Any mammal can carry a strain of the virus and pass on, by way of saliva contaminated with the pathogen, the infirmity to a human being. It is not necessary that the person be bitten by a sick animal. Sometimes, it is enough to have been in contact with the skin or the mucus of the individual with the saliva of a sick animal. “But the risk of acquiring rabies is 50 times greater by way of a bite than by a scratch”, advised Dr. Neuza. In practice, the main propagators of rabies in the urban environment are cats and dogs, and in the rural areas, bats that feed on blood.

Since the rabies virus has a long incubation period in human beings, in general from one to two months, the anti-rabies vaccine should be applied as soon as the contact with a potentially infected animal has taken place. It works as if it were a remedy after having been contaminated. The most common scheme for immunization prescribes five doses of the vaccine, applied over a 28 day period. As well as the people who have been in contact with the suspected infectious animals, professionals who present a higher risk of contracting rabies, such as veterinarians and zoo technicians, should take the vaccine in a preventative manner.

Responsible for up to 70,000 deaths per year in the world, human rabies is under control in Brazil, especially in urban centers. Historically, the number of cases of the illness has shown a tendency to fall. At the start of the decade of the 1980’s, rabies had annually killed more than 160 people in the country. Twenty years later, the number of deaths circulates at around ten per year. “But, as we’re dealing with a zoonosis, it’s impossible to eradicate it completely”, commented the medical doctor Wagner Augusto Costa, from the Pasteur Institute, who coordinated the tests on human beings with the new vaccine from the Butantan Institute. Last year the number of deaths again went up, although at levels less alarming than in past years. There were around thirty deaths, two thirds of which came from outbreaks in the state of Para of rabies contracted through bats. These flying mammals take the place of cats and dogs as the principal vector of the illness among Brazilians. During this current year the situation is repeating itself. From January until July of 2005, the illness has killed 15 people in Para and three in the state of Maranhao. All of them having contracted rabies through bats. “Deforestation is driving the wild bats towards small towns, increasing the risk of the transmission of the illness to human beings”, affirmed Dr. Neuza.

For cats and dogs
The new anti-rabies vaccine for veterinary use developed at the Butantan Institute is not as pure as the human version of the product. But it will be more efficient and cost less than the product used today in the public program for immunizing cats and dogs, manufactured from the brain of recently born mice. The new vaccine, whose production process technique will be transferred to a company in São Paulo, is manufactured from a type of renal cell of the hamster denominated BHK, which needs a culture medium with serum in order to grow. Nevertheless, this culture material, coming from cellular lineages established years ago and its process of reproduction in the laboratory, has already been dominated by the Butantan Institute. Or that is to say, it is not necessary to kill new animals in order to obtain more BHK cells.

In order to manufacture the annual 33 million doses of anti-rabies vaccine for animal use that is provided by the National Program of Prophylaxis of Rabies, from the Ministry of Health, the Technology Institute of Parana (Tecpar) every week sacrifices 120,000 recently born mice. This is because the company has a need for rodent nerve tissue to be used in the manufacture of the vaccine’s formula “The vaccine manufactured via BHK cells is going to avoid the deaths of these animals”, says Neuza Maria Frazatti Gallina, from the Butantan Institute. Tests carried out on cats and dogs at the São Paulo State University (Unesp), of Araçatuba, and in cattle by way of the West São Paulo University (Unoeste), in the town of Presidente Prudente, show that the vaccine from the Butantan Institute is efficient in conferring immunity to animals.

The project
The study of humoral and cellular immunity induced by vaccine against rabies in Vero cells; Modality Regular Line of Assistance for Research Project; Coordinator Neuza Maria Frazatti Gallina –  Butantan Institute;
Investment US$ 85, 000.00 (FAPESP)