Heterologous synthesis and purification of a non-toxic homologue of diphtheria toxin, aiming for applicability as an antigen in the selection of monoclonal antibodies

• Grant number: 24/03108-1
• Support Opportunities: Scholarships in Brazil - Master
• Start date: April 01, 2024
• End date: March 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Ana Maria Moro
• Grantee: Ednilson Donisete de França Junior
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 20/07040-1 - Center of Research and Development in Immunobiologicals, AP.NPOP

Abstract

Diphtheria is a fatal disease caused by infection with Corynebacterium diphtheriae, a bacterium that, when infected by a corynebacteriophage, begins to carry the tox gene sequence, which encodes the diphtheria toxin. Individuals affected by the disease exhibit cell death at the sites of toxin production, with characteristic lesions in the pharynx and skin. In more severe cases, where there is hematogenous dissemination occurs, damage to the nervous and cardiovascular systems may arise. Diphtheria toxin has 535 amino acid residues in its mature form, with two polypeptide subunits. The B subunit has an R receptor binding domain, and the interaction of the domain with cell surface receptors results in the endocytosis of the interaction complex by the target cell. The transmembrane domain present in the toxin and the changes resulting from acidic conditions in the endosome allow the transposition of A subunit into the cytosol. In the cytosol, A subunit, which features the catalytic domain C, transfers ADP-ribose to elongation factor EF-2, preventing the ribosome from continuing the translation process, culminating in cell death. The most efficient method for combat diphtheria is vaccination using toxoid, however, there are reports in several countries, including Brazil, of a reduction in vaccine coverage. The disease frequently affects areas with precarious socioeconomic conditions, high population density and low vaccination coverage, resulting in an increase in the number of cases and outbreaks in recent years. In 2021, in an epidemiological update issued by the World Health Organization (WHO) and the Pan American Health Organization (PAHO), members countries were emphasized on the need for a plan to maintain permanent supplies of diphtheria antitoxin, seeking the capacity to deal with potential disease outbreaks. The treatment with equine-derived diphtheria antitoxin (DAT) is currently the only form of therapy, administered alongside auxiliary antibiotic medication. However, it is acknowledged that equine serum has a limited global supply, and the treatment is subject to serious allergic reactions. Diphtheria is an important disease to contain globally, gaining attention due to disruptions in health infrastructure, lack of accessibility to vaccination resources, increased longevity, neglect and other issues that render populations susceptible to outbreaks of the disease. Knowing the deficiencies in vaccination coverage, the increase in case records, the demand for diphtheria antitoxin serum, disease containment policies, and the limitation of the use of animals, the search for human monoclonal antibodies (mAbs) is a viable and promising alternative. One of the most critical factors for antibody selection is obtaining a well-characterized, homogeneous antigen in unlimited quantities, which represents the objective of this project.