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Coming soon: the vaccine four-in-one

A cocktail immunizes animals against tuberculosis, diphtheria, tetanus and whooping cough

For the first time, among the many attempts going on throughout the world, Brazilian scientists were able to express in a microorganism proteins made from the bacteria that provoke diphtheria, tetanus, and whooping cough, thus opening the way for a vaccine against all four illnesses. This was done on the microbacterium BCG, used as a vaccine against tuberculosis.

“We have managed to produce an immune response in mice that protected the vaccinated animals.” reveals the chemist Dr. Luciana Cerqueira Leite, coordinator of the project developed at the Butantan Institute. She did her post doctorate at the Pasteur Institute in France, where she studied the possibility of making a vaccine against HIV (the virus that provokes Aids) based on recombined BCG and from where she brought some of the tools used in the development of the quadruple vaccine.

The BCG (bacillus of Calmette-Guerin) is a result of the work of the weakening of Mycobacterium bovis, that Albert Calmette (1863-1933) and Camille Guerin (1872-1961) worked on for 13 years at the Pasteur Institute at the beginning of the 20th century. After tests with animals and the action against after infections was proved, both for M. bovis and for M. tuberculosis, BCG was used for the first time as a vaccine in 1921. In 1927, the League of Nations recommended its generalized application, and today it is the vaccine most widely used in the world.

Recombined BCG
Extremely safe, – the slight side effects don’t put the patient’s life at risk -, BCG has qualities that serve as a base for another vaccine. It is a microbacterium whose walls have components that give it a very high resistance. A single dose of this living vaccine, given to newly born babies, stimulates the immune system for around 20 to 30 years. Also, it is easy to produce and is of low cost, around US$ 0.08 per dose.

Nevertheless, when Luciana and her colleagues at the Biotechnology Center of Butantan, at the Pasteur Institute and at the Italian laboratory of Biocine, began their work in 1997, the molecular biology of BCG had still not been well established. For this reason, the time to make recombined BCG, or that is to say, to manage to make the BCG express the proteins of the other three agents, those that provoke diphtheria, tetanus and the whooping cough, was greaterthan if it had been carried out through a better known microorganism such as Escherichia coli. “The first recombined BCG was made in 1990, but over the last few years there has been a lot of advance in this area.” says the researcher. Called recombined BCG – DPT, the quadruple vaccine is formed by micro bacteria of BCG containing non-toxic pieces of the proteins of the bacteria that provoke the other three illnesses, diphtheria (the D of the acronym) whooping cough or pertussis (P) and tetanus (T). To make the BCG produce these proteins it was necessary to use expression vectors, the tools that Luciana brought back from the Pasteur Institute of Paris.

Expression vectors are pieces of circular DNA called plasmids and contain all that is necessary to make BCG produce proteins of other pathogens (agents that cause disease). These proteins are the antigens that in the body of the vaccinated person induce the immune response to the organism, for example, the production of antibodies. “The plasmids are made from pieces of DNA of various micro organisms, and the selection of these pieces of DNA is done in accordance with the necessity of each experiment. Each one constructs a plasmid as is needed.” she explains.

In this way, the BCG micro bacteria of the quadruple vaccine receive a non-toxic piece of toxin of tetanus, of the toxin of diphtheria with a mutations that turns themselves inactive, as well as two antigens of whooping cough. One of these antigens is the pertussis toxin, with a mutation that makes it non-toxin, and the other is a non-toxic fragment of haemagglutinin, a component of the bacterium that helps to provoke its linking to the organism. Luciana explains that the bacterium has to colonize the organism in order to begin to grow and to be toxic. If in the vaccine a component that assists adhesion is placed onto it, the organism is then going to produce in response an antibody against this adhesion, that impedes the bacterium from colonizing the immunized body.

Patented genes
Two genes used in the research, the antigen of diphtheria and that of the whooping cough, came from the Italian research laboratory Biocine, associated with the North American pharmaceutical company Chiron. The genes are the property of this association, and if the vaccine proves to be efficient and safe in humans, it will be necessary to discuss patents.For Luciana, this should not stop the studies. “The discussion about patents is world matter, very controversial and it is not closed yet. Besides this, the philosophy of the project is to create a vaccine that makes the process of immunization easier. With only one vaccine, applied to newly born babies at a moment when nearly all women, even in the farthest and most rural areas of the country, have access to a hospital or health center at the time of child birth, we can immunize the children against four illnesses.” The measure would generate an economy in the immense cost of the vaccination campaigns and would avoid the re-enforcement doses, something which the rural population and those with large families don’t always manage to comply.

The researcher sums up the proposals in a sentence: “The attempt with BCG-DPT is to make an effective and cheap vaccine, with fewer side effects, that reaches a greater proportion of the population.” She explains that the component against the whooping cough is a little reactogenic (it provokes a local reaction) and that there is already in the developed countries another type of vaccine against whooping cough, though it is much more expensive than the one used in Brazil. The vaccine of recombined BCG will be safer and will not have an additional cost.

Eliminating resistance
Having finished the first stage successfully, that is having expressed on the BCG antigens of other bacteria and having produced an immune response in mice, the next stage will be to verify if the vaccine continues to be efficient against tuberculosis itself, as it is expected. To verify this, they are already running tests, together with researchers of the School of Medicine of Ribeirão Preto of the University of São Paulo.

Another task is to eliminate from recombined BCG the genes resistant to antibiotics, whose presence in a vaccine always brings the danger that one day they will transfer this resistance to another invading organism. In the laboratory set up for the project in Butantan, they are already looking for a technique to wipe out these genes. The laboratory has biosecurity of level II, required by the National Technical Commission of Biosecurity (CTNBio) to work with this type of bacterium, which has a relative degree of risk. The risk of tuberculosis, for example, is higher. The Butantan Institute is responsible for the premises while FAPESP pays for the air conditioning system and the equipment.

New projects
As well as attempting to eliminate the resistant genes within the Butantan Institute itself, with the collaboration of a researcher form the University of Pelotas (RS), Luciana keeps in touch with a New York laboratory where a system of recombined BCG without the gene resistant to the antibiotic is working.

Once these stages have been overcome, there will be new tests on mice and guinea pigs, later on possibly on monkeys, until they get to human beings. Only then will the vaccine be ready for production. The process is long and requires various years of research. For this reason Luciana is already dealing with two new projects. A thematic project that includes a vaccine of recombined BCG against pneumonia, is also being financed by FAPESP, and another of a veterinary vaccine that is a project for the European Union. She believes that the tools developed in these new studies could quicken the conclusion of the quadruple vaccine.

The Project
Development of Vaccines of Recombined BCG – DPT  (nº 96/11539-0); Modality Regular line of research assistance; Coordinator Dr. Luciana Cezar de Cerqueira Leite – Butantan Institute of the Secretary of health of the State of São Paulo; Investment R$ 124,000.00 and US$ 55,000.00