Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay.

Salvatore Giovanni De-Simone,Salvatore Giovanni De-Simone,Jorge Soares De Pina,Guilherme Curty Lechuga,Flavio Rocha Da Silva,Paloma Napoleão-Pêgo,Larissa Rodrigues Gomes

doi:10.3390/vaccines9040313

Salvatore Giovanni De-Simone, Salvatore Giovanni De-Simone + Show 5 more

Open Access

https://doi.org/10.3390/vaccines9040313

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Journal: Vaccines	Publication Date: Mar 26, 2021
Citations: 14	License type: CC BY 4.0

Affiliation: Fundação Oswaldo Cruz, Universidade Federal Fluminense

Abstract

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

Highlights

Diphtheria is a vaccine-preventable, bacterial disease caused by toxin-producing strains of Corynebacteria diphtheriae and is once again a growing concern
In this work, using a more refined methodology of epitope synthesis, we report the mapping of B-cell continuous epitopes of diphtheria toxin (DTx), the topological characterization of the epitopes, and the production by chemical synthesis of DTx-bi-epitope and its use as antigen for diphtheria diagnosis
The epitopes in the DTx (560 aa) were identified based on recognition of peptides in a synthesized library by mice antibodies immunized with DTx

Summary

Introduction

Diphtheria is a vaccine-preventable, bacterial disease caused by toxin-producing strains of Corynebacteria diphtheriae and is once again a growing concern. It is endemic in Asia (India, Indonesia, Iran, Nepal, Pakistan, etc.), Africa (Ghana), and some countries of South America [1,2,3] but well-controlled in countries with high vaccination coverage. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays

Methods

Results

Conclusion