Mapeamento dos epitopos da toxina épsilon de Clostridium perfringens tipo D e produção de imunógenos de peptídeos sintéticos

Abstract

Clostridium perfringens type D epsilon toxin (ETX) is the main responsible for domestic ruminants enterotoxemias. Due to its high toxicity, it is now considered to be a potential agent of bioterrorism or biowarfare. There are currently no vaccines or treatments against the effects of this toxin in humans. In contrast, vaccination is the primary control measure of enterotoxemia in ruminants. Although commercial immunogens can be effective in preventing enterotoxemia, their quality varies greatly between countries and manufacturers. Thus, researches in immunology and vaccinology that support the development of new vaccines and treatments against ETX in domestic ruminants and humans are imperative. In light of this, the objective of the present study was to map the epitopes of C. perfringens type D ETX and to produce immunogens containing synthetic peptides. One hundred thirty overlapping 15-mer peptides frameshifted by three residues corresponding to ETX sequence were prepared in a cellulose membrane by using the Spot technique. Sheep and rabbit sera against purified ETX were used in immunochemical assays to test the interaction between anti-ETX antibodies and the synthesized peptides. Based on the reactivity results of these assays and on physicochemical characteristics, six mapped epitopes were separately employed to immunize mice. Peptides containing the primary structure of these six epitopes were synthesized by solid phase synthesis, encapsulated in liposomes and aluminum hydroxide was added. Six groups of six mice each received four doses of the respective immunogens spaced by 10 days. At the end of this process, the collected sera were titrated by competitive ELISA. Based on the results of the immunochemical assays, a total of 16 probable epitopes were identified in the primary structure of ETX. This result is consistent with immunological mechanisms of immune response against proteic antigens, which are processed to several smaller peptides ranging the entire protein structure, and are then presented to CD4+ T lymphocytes. Three out of the six antigenic determinants used in the immunizations induced antibodies that were detected by a competitive ELISA, and are probably immunodominant epitopes. The inhibition rates were 4.35, 9.95 and 7.68% for the epitopes number 3, 4 and 16, respectively. The determinants number 3 and 4 are partially overlapped, linear and spatially close to each other, and also belong to ETX domain I. This region is likely to be involved in the binding of ETX to its cellular receptors. It also has amino acids essential to the protein-receptor interaction and thus essential to the toxin cytotoxicity. Epitope 16 is part of the carboxy-terminal portion of ETX, and belongs to domain III of the protein. This region seems to be involved in the oligomerization of ETX, phase that precedes cell pore formation. The results of the present study provide valuable information for understanding the structural, pathogenic and immunological characteristics of ETX, and can thus assist in the development of new vaccines and therapies against the deleterious effects of the toxin in animals and humans.