The material is ready. Sequences of bacterium proteins responsible for a reaction by the organism that unleashes rheumatic fever will start to be tested in the laboratory and on experimental animals. They will serve as a basis for a vaccine against the throat infections caused by the microorganism that originates this serious disease. Isolating the sequences of proteins is the fuel for the take-off of a recently approved project under the Partnership for Technological Innovation (PITE) program and coordinated by Luiza Guilherme Guglielmi and Jorge Kalil, both from the Heart Institute (Incor) of the University of São Paulo (USP).
With investments of US$ 3 million, in equal proportions from FAPESP and from the Laboratório Teuto-Brasileiro, a Brazilian pharmaceutical industry headquartered in Anápolis (GO), which produces generic medicines, the vaccine project aims at the solution of an illness that in its most serious form attacks the cardiac tissue, destroys the mitral and aortic valves, affects predominantly the poor populations, and is a burden in the expenditure of the public health systems. The fatal bacterium, Streptococcus pyogenes, also known as a Group A streptococcus, in the sights of the future vaccine, has been the subject of 12 years of study by the team from Incor’s Immunology Laboratory, run by Kalil.
It does not directly cause the disease, but it triggers off a process that makes the organism turn against itself – a characteristic of the so-called autoimmune diseases, like rheumatic fever. Without treatment, what begins as a simple tonsillitis caused by the streptococcus evolves to feverish states, joints, pain, and finally an attack on the heart tissue. At each occurrence of the disease, the picture is repeated. When parts of the cardiac tissue have already been destroyed, the patient can only be saved by surgery – usually replacing the mitral or aortic valves, at a high cost and with possible permanent damage.
In 1998, 90% of children’s heart surgeries carried out in Brazil were due to this fever. With adults, the percentage comes to 30%, still considered high. There is a total of 10,000 surgeries a year, at a cost of from US$ 8,000 to US$ 10,000 each. “Rheumatic fever is a very expensive disease for the health system of any country”, says Luiza. In Brazil, where 18,000 new cases are recorded every year, a rheumatic fever patient with heart problems costs US$ 10,000 a year to the Single Health System (SUS, the public health system in Brazil).
The destructive reaction of the organism has its logic. Kalil draws a good-humored analogy with the investigations in the recent kidnapping of advertiser Washington Olivetto, in which Chilean citizens were involved. Suppose that there were a system for tracking telephone calls capable of identifying languages, though not accents: without the imprisonment of the Chileans, all Spanish-speaking people would be potential suspects. In the case of rheumatic fever, there are proteins in the cardiac tissue that remarkably similar to those of the bacterium.
The similarity makes the cells of the immunological system attack, not the bacterium, but the heart, through a reaction called biological mimesis. The organism establishes the lethal identification: it starts to attack not only the bacterium, but the tissue as well. As this confusion may arise, it is crucial to block the advancement of the process. This is normally done with a swift treatment of throat infections with an antibiotic – not always possible in regions poor in health services.
The solution – radical and definitive – should be to avoid the bacterium attack through a vaccine capable of immunizing all children, the main victims of the disease. Rheumatic fever generally attacks the age group from 5 to 18 years, predominantly in poor countries, and it hits globally between 3 and 4% of those infected and untreated. Among these, from 30 to 45%, probably due to a genetic predisposition, develop the severest form of the disease, which attacks the heart.
The immune system attacks the tissue from four to eight weeks after the beginning of the throat infection, which usually lasts from seven to ten days, when there are no more bacteria to attack. Normally, the system combats the bacterium by activating the blood cells called white globules or leukocytes (above all T and B lymphocytes), responsible for the destruction of the streptococci.
In genetically susceptible children, what happens is an aggression against their own tissue, the so-called autoimmune reaction. The immunological system stores in a sort of memory the structure of the bacterium, which allows it to be recognized, should it re-appear. In these children, it recognizes structures – pieces of proteins – of the heart, similar to those of the bacterium. In a mechanism known as cross reaction, these structures come under attack. “This sort of ‘biological error’ means that auto-immune diseases are much studied”, Kalil points out.
The team’s path towards the conception of the vaccine was drawn up from the streptococcus cellular wall. Luiza explains that the bacterium has an external protein in the cell wall, called M protein. This is the protein that sets off the cross reaction between the proteins of the heart and those of the bacteria, both with similar structures. But this same protein has regions that do not set off these cross-reactions, and apparently provide protection against the bacterium. “We studied the two regions, those that set off reactions against the heart and those that do not”, the researcher comments.
The team from Incor intends to make a vaccine from the regions that set off, not the cross-reaction, but a protective response. There are three distinct approaches. “The ‘good’ sequence may have combinations of peptides (protein fragments) to make up the vaccine”, says Luiza. “Or we can construct a recombinant protein, obtained by means of an innocuous bacterium, that contains these sequences”. The third option, the least probable, according to Kalil, is the vaccine from DNA (deoxyribonucleic acid, the carrier of the genetic code), by means of a vector. “This possibility has been developed conceptually, but not technologically”, he says. “There is still no DNA vaccine in use”.
The researchers are at the stage of choosing the regions of the proteins. “We have to start the peptides synthesis from these regions, develop the recombinant protein, and implant the methodology for a possible DNA vaccine”, Luiza comments. In technological terms, the group from Incor proposes a vaccine either with a fraction of protein, or with the DNA left bare without the risk of setting off the disease.
In any of the approaches, the vaccine will obviously be tested on animal models before reaching human beings. Mice of two kinds will be used: those not genetically manipulated (isogenics), and the manipulated ones (transgenics). Those from the second group will contain a human gene that acts in the recognition of the vaccine and is extremely important for setting off the response of the immune system. “We want to get as close as possible to what would be the response in human beings”, Luiza explains. This is the first stage. In the following stage, the researchers will move on to testing with primates, and, later on, with human beings. They do not yet have a primate model. The rat model, in fact, the Lewis rat, was only found at the end of 2001, for developing heart disease after rheumatic fever.
The experiments with animals will last about five years. Afterwards, there will be another two to three years with clinical tests on human beings. The range of action of the vaccine does not cover just the prevention of rheumatic fever. Luiza stresses that this is the fundamental point, but the medicine that should emerge from the research will have a far wider ranging action. First of all, it will prevent, in general terms, infections from type A streptococcus, like the one that causes the throat infection, common in children: a recent study carried out in nurseries in Curitiba showed that 24.5% of the children had streptococcus in their throats. “As we do not know who is going to develop the disease”, Kalil notes, “the vaccine has to protect them all”.
Another advantage will be reducing the antibiotics dosage in patients with rheumatic fever. In principle, the vaccine will be enough to prevent further infections more permanently in children who already have the disease and are obliged to take penicillin (usually benzatine) every 21 days. This period of medication with the antibiotic is based on the cycle for developing a throat infection from streptococcus, which, after all, is roaming all over the place. “We are all liable to this infection, which has peaks when it occurs most: fall, winter, and the beginning of spring”, the researcher recalls.
The problem is that with each new infection caused by streptococcus in children who have already developed the infection, the problems come back – fever, pain in the joints, and cardiac damage. In milder cases, the heart is not affected, but patients may develop chorea, a neurological disturbance that sets off involuntary movements and which is usually mistaken for the so-called St. Vitus’s disease. In these cases, the diagnosis is made early, because the problem is very visible, which is not the case when there is a heart condition.
The researchers from Incor stress the social dimension of the problem. “The majority of the children affected”, Luiza notes, “have low income and sometimes live where there is no health post, or where access to one is difficult”. For these reasons, she recalls, the number of children that do not stick to the treatment is high.
Kalil adds: “There is the social problem, the economic one, and the public health problem. It is a disease that affects most the poor populations, because the rich probably treat the throat infection earlier. And there is the inherent risk with developing the vaccine, the challenge of getting a vaccine that produces a good effect, without the risk of developing the disease, since the same protein connected with the protective response may unleash the disease”. In short, the vaccine cannot cause any disease, not even the throat infection.
The objective is to have the vaccine listed amongst the obligatory ones, which every child should take at a certain age. “We still have to assess this age, but it will certainly not be before the age of three, because there are vaccines which very young do not respond well to, above all those made from proteins”, the researcher says. Twelve years ago, the group started to identify factors for susceptibility to rheumatic fever in patients at USP’s Hospital das Clinicas. Next, they studied how auto-immunity – the attack on the patient’s own organism – is triggered off, and in 1995 published an article on the subject in Circulation, the top scientific publication in cardiology. The magazine’s editorial regarded the group’s findings a landmark in the understanding of this disease.
In these years of work, the group has conquered other marks of recognition. One of them was the 1997 Unibanco Health Prize. The researchers attribute their results to their insistence on an intensive study of the description of the disease. Luiza tells how she has never forgotten the closure of the Vaccine of the Future Congress, in October 2001, at the Pasteur Institute in Paris, when the comment was made that, in spite of advancement by study groups from all over the world, there was still no formula for a vaccine, but that those who a better knowledge of the disease to be treated will find the best option.
The researchers are also supported by cutting edge equipment and a laboratory that has expanded into an area of 750 square meters. A special part of the project involves studying the proteome. “The more we study proteins, the more we will avoid a crossed reaction, and the safer the vaccine will be”, Luiza concludes. Kalil says that one of the major issues not resolved by science, mentioned in a balance drawn up at the end of the 20th century, is knowing how an auto-immune disease is unleashed.
The agreement between Incor and the company provides for FAPESP receiving 3% of net sales (gross sales less taxes) generated from the marketing of the vaccine.
Development of a Vaccine against the Group A Beta Hemolytic Streptococcus (nº 00/14549-4); Modality Partnership for Technological Innovation; Coordinators Luiza Guilherme Guglielmi and Jorge Elias Kalil Filho – Heart Institute/USP; Investment US$ 1,5 million