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immunology

Mycosis from the earth

Research advance into a vaccine and target genes to fight a fungus typical of South America

After almost 20 years of work, researchers from the Federal University of São Paulo (Unifesp) have arrived at a potential vaccine for the Paracoccidioides brasiliensis which causes paracoccidioidomycosis, a mycosis typical of Latin America that affects 10 million individuals (80% of them in Brazil). Tests on mice and with blood samples from human beings revealed that a mixture of fragments of a protein extracted from the fungus itself generates an immune response in 75% of the cases, very close to the level desirable for a vaccine. With new tests, which should take a few years, this vaccine ought to become an effective alternative for preventing the disease and increasing the efficiency of the usual treatments – on the basis of medicines of the sulfa kind and fungicides -, which can extend for up to five years.

Another line of work that may result in news ways of fighting the disease is the study of the P. Brasiliensis genome. In an independent but complementary manner, two groups, one from the Ribeirão Preto School of Pharmaceutical Sciences of the University of São Paulo (USP) and another made up of researchers from the center-west region – sequenced 11,000 genes of the fungus. They found 40 genes that represent future targets for medicines, as they are ssential for the survival of the parasite in the human organism: they are linked to the aggressiveness of the fungus, with its capacity for adhering to the cells of the host organism (for example, those in the lungs), or with the transformation of the mycelium, multicellular filaments that contaminate living beings, in yeast, cells that spread over the body.

This mycosis causes sores on the skin and lesions in the mouth, and can contaminate the lungs and infiltrate the bones, the joints and the central nervous system. It is spreading, above all, among rural workers in recently occupied areas, such as the states of Rondônia, Tocantins, Pará, Mato Grosso and Acre. It is probably a consequence of the deforestation and preparation of the soil for planting, which increases the number of particles of the fungus in suspension in the air.

The researchers from Unifesp extracted from the fungus itself the ingredients of what, in experiments with mice and human blood, proved to be a possible vaccine against paracoccidioidomycosis. The team under the coordination of Luiz Rodolpho Travassos found that small fragments of a protein (peptides) from the surface of the fungus, glycoprotein 43, or gp43, are capable of stimulating the response of the defense system of rodents against P. brasiliensis, a good indication of what may happen in the human organism.

While she was still studying for a doctorate under Travassos, Rosana Puccia had discovered in 1986 that gp43 activated the human immune system against the fungus – it would work as an antigen, as they researchers say. The entire molecule, besides stimulating the proliferation of t-lymphocytes, a kind of defense cell, would set off the production of antibodies, which is not effective in the case of this mycosis. The idea then arose of looking for a stretch of the gp43 to generate only the benefic effect: the production of t-lymphocytes.

P10, the star
Some time later, the researchers found that a small part of gp43 would also awaken the immune response – it was a segment made up of only 15 amino acids (the blocks that make up proteins) known by the abbreviation P10. In tests with three strains of mice, P10 stimulated the proliferation of t-lymphocytes against the fungus in all the animals. After immunizing these rodents with the peptide, Carlos Taborda, who is also studying for a doctorate under Travassos, injected into the animals’ windpipe a suspension containing an aggressive form of P. Brasiliensis and observed that there was an effective protection against infection: the lungs suffered no damage and the fungus did not disseminate itself through the organism. “P10 is our great star”, says Travassos.

It was still necessary to discover if P10 – or other peptides of gp43 – would work in man, like a key that fits a sort of chemical lock, the class II molecule MHC (which stands for major histocompatibility complex). Made up of two coupled proteins, this complex recognizes molecules that are foreign to the organism and sets off the alarm of the defense system. In order to create the protection desired in genetically distinct populations, P10 would have to be what researchers call a promiscuous antigen – promiscuous meaning binding itself to the largest possible number of the some 300 types of MHC class II molecules identified in the human being.

To find out if this would happen, Travassos worked with researchers from USP’s School of Medicine and a computer program that analyzed the compatibility between the structures of P10 and those of 25 of the 300 kinds of class II MHC. It worked out: P10 fitted, with a high degree of affinity, 22 of the 25 kinds of class II MHC analyzed – another four fragments of gp43 showed similar potential. It may not seem much, but these 25 are the histocompatibility complexes shown by 90% of the Caucasoid population, the largest ethnic division of the human species, which includes the peoples native to Europe, northern Africa, southwestern Asia, including India, with characteristics like the color of the skin, varying from light to olive, and hair, from straight to wavy and frizzy.

It remained to be found out how the five molecules identified would behave in contact with cells from the human defense system. This time, it was Leo Kei Iwai, from USP, who tested the peptides in blood samples taken from 29 people who had been given treatment against infection by P. brasiliensis and still showed an immune reaction against the fungus. A complex picture appeared: although it was still the most effective antigen, P10 activated t-lymphocytes specific to the antigen in about half of the samples examined – a success rate that falls short of that required for the production of a vaccine with a single peptide.

The good news is that the other peptides worked when P10 failed. According to the most recent results, the five mixed antigens show a performance of 75%. “This level is now ethically adequate for thinking about a vaccine”, says Travassos. Even so, it is still necessary to overcome other stages before getting a viable vaccine for the first tests on human beings. The first of them is to produce the peptides in a high quantity and level of purity, besides discovering which is the most efficient formulation for immunization.

Other targets
In parallel, Gustavo Goldman’s team, from USP in Ribeirão Preto, sequenced almost 5,000 genes of P. brasiliensis. At the same time, the regional Center-West Genome network, a consortium made up of 13 institutions from the states of Goiás, Mato Grosso, Mato Grosso do Sul and from the Federal District, mapped almost 6,000 genes, and in the February issue of Yeast published a list of the 18 most active genes of this fungus. According to the coordinator of this group, Maria Sueli Soares Felipe, from UnB, after discounting the possible repeated genes, the team from São Paulo and the one from the Center-West have mapped almost the whole of the fungus’s genome, estimated at 15,000 genes.

Goldman compared the sequences from P. brasiliensis with 60 genes associated with the capacity for causing disease – or the pathogenicity – of Candida albicans, a fungus that causes candidiasis, a common mycosis that gives rise to whitish sores in the mouth and reddish blotches on the skin. The results: P. brasiliensis shows 26 genes similar to those of C. albicans, as is attested by an article published in February’s Eukaryotic Cell. “These are the possible targets to be combated.”

The team from the Center-West has managed to genetically alter yeasts of P. brasiliensis, adding a gene to its genetic material by means of a technique called electroporation, which opens pores in the cell walls of the fungus and allows the entry of foreign genetic material – a procedure that is essential for deactivating genes. The group arrived at the genetic transformation of five to ten funguses per microgram of DNA and is working to increase this performance, so as to facilitate the experiments. “What we have already managed is a good start”, says Maria Sueli. “It is a sign that it is possible to alter the fungus genetically.”

The projects
1. Molecular Biology and Immunobiology of Purified Exocellular Components of Paracoccidioides brasiliensis (nº 95/00559-8); Modality
Thematic project; Coordinator Luiz Rodolpho Travassos – Unifesp; Investment R$ 284,832.98 (FAPESP)
2. Molecular Characterization of Genes Involved in the Process of Pathogenicity and Virulence of Paracoccidioides brasiliensis (nº02/08711-9); Modality Thematic project; Coordinator Gustavo Henrique Goldman – USP; Investment R$ 554,380.00 (FAPESP)
3. Functional and Differential Genome of Paracoccidioides brasiliensis; Modality Center-West Genome Network; Coordinator Maria Sueli Soares Felipe – IB/UnB; Investment R$ 1,430,000.00 (MCT/CNPq)

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