Expressão, caracterização e avaliação funcional de anticorpos humanos antitetânicos

Author(s)

Daniela Yumi Takata

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-09-30

Examining board members

Ana Maria Moro; Renato Mancini Astray; Marcelo Mendonça; Sergio Daishi Sasaki; Mari Cleide Sogayar; Marco Antonio Stephano

• Advisor: Ana Maria Moro

Abstract

Tetanus is a neuromuscular disease caused exclusively by the action of the exotoxin produced by Clostridium tetani (C. tetani). Despite there is a safe and low-cost vaccine, the incidence and mortality rate of tetanus remain worrying. Therefore, in cases at risk of tetanus, passive immunization with equine anti-tetanus serum (ATS) or hyperimmune human tetanus immunoglobulin (TIG) is required. In search of therapy based on modern biotechnology, our group previously obtained human anti-tetanus monoclonal antibodies (mAbs) derived from B lymphocytes isolated by single cell sorting. The objective of this work was to expand and evaluate the panel of anti-tetanus mAbs, for the proposal of candidates for future use in therapy. The panel of mAbs was tested by ELISA, Western Blot, and tetanus toxin (TeNT) binding to ganglioside GT1b inhibition assay. Five mAbs were analyzed by TeNT peptide microarrays to identify their epitopes and by in vivo neutralization assay, individually or associated in two or three. Only when a mixture of three mAbs that bind to different domains of TeNT was neutralizing, demonstrating the importance of synergism for the neutralizing action. In order to reduce animal testing, some alternatives were performed in an attempt to screen the antibodies to be tested in vivo. However, the execution of the tests was limited by the low concentration of expressed mAbs and availability of TeNT, besides other assays limitations. Due to the difficulty in analyzing a large panel of antibodies, we decided to focus on obtaining permanent cell lines to produce the mAbs that neutralized the TeNT in vivo. We ordered the synthesis of vectors containing the sequences of the constant regions of human heavy gamma 1 and light kappa chains. We also purchased vectors with the optimized sequences of the variable regions of two mAbs in order to compare with the sequences generated in the laboratory. There was a difference between the antibodies yield obtained with the optimized sequences and original sequences. However, as expected, the optimization of the sequences did not change the function of the mAbs. (AU)

• FAPESP's process: 16/08782-6 - Neutralizing human monoclonal antibodies
• Grantee: Daniela Yumi Takata
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)