Immunogenic activity by Amblyomin-X in human t lymphocytes

• Grant number: 14/23493-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): March 01, 2015
• Effective date (End): August 31, 2015
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Ana Marisa Chudzinski-Tavassi
• Grantee: Jean Gabriel de Souza
• Supervisor: Bernard Maillere
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Research place: Commissariat à l'énergie atomique et aux énergies alternatives (CEA), France
• Associated to the scholarship: 12/06944-8 - Effect of Amblyomin-X at interface of the immune system and hemostasis during progression RENCA tumor (murine renal cell carcinoma): in vivo and in vitro, BP.DR

Abstract

Animal toxins, derived from snakes, spiders, insects and snails of the genus Conus, are remarkable collection of molecules with a pharmaceutical potential. These toxins represent a great diversity of functions and are involved in the evolution of several natural process. They interact, with high affinity and selectivity, with molecular targets that play critical physiological roles. Due to their high selectivity for molecules involved in some diseases, many of these toxins were successfully tested in preclinical models, while others are already used for the treatment of diseases in humans. The potential ability of therapeutic proteins to induce immunogenicity in the host, represents an important issue in their development. The Antibodies produced in response to a given molecule with new drug potential, may be capable to neutralize and cancel its therapeutic effect. CD4 + T lymphocytes are the main cells that can trigger the immune response to a particular molecule with therapeutic potential. Recent advances in the mechanisms of immunogenicity have shown that the evaluation of potential drugs recognition by CD4-T is very important to predict their immunogenicity in humans. The Butantan found small proteins originally from animals, such as Amblyomin-X and Lopap. Amblyomin-X is a recombinant protein with Kunitz domains isolated from a cDNA library of salivary glands of Amblyomma cajennense tick. It inhibits factor Xa activity and exerts a cytotoxic effect on cancer cells by inducing apoptosis. Lopap is a recombinant protein composed of 180 amino acids, with a lipocalin structure, isolated from the Lonomia obliqua caterpillar bristles. Lopap has an anti-apoptotic activity and is involved the prothrombin activation and renewal of the extracellular matrix. A recent approach developed by CEA will be applied in both molecules discovered by the Butantan Institute to test the risk of their immunogenicity. Amblyomin-X and the Lopap will be subjected to human T cells amplification assay , which has been developed by the CEA in an attempt to detect the blood T cells of normal individuals, that respond to these molecules. Briefly, healthy individuals will be selected based on their HLA. CD4 lymphocytes will be purified and co-cultured in various repetitions of autologous dendritic cells previously sensitized with Amblyomin-X or the Lopap. After a few weekly passages of antigenic stimulation, T cells will be amplified and evaluated for their specificity to these molecules by ELISPOT. The frequency of the tested molecules to specific pre-existing T cells will be calculated based on the number of wells culture that reacted to the proteins. The risk of immunogenicity will be evaluated based in this frequency. To detail response of T cells origin, the immunogenic sequences present in the Amblyomin-X and Lopap will be identified using synthetic peptides for each epitope. (AU)