Development of a novel multiplex electrochemiluminescent-based immunoassay to aid enterotoxigenic Escherichia coli vaccine development and evaluations

Subhra Chakraborty,Jessica Brubaker,Clayton Harro,Thomas Weirzba,David Sack

doi:10.1016/j.jim.2019.04.003

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.

Highlights

Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of morbidity and mortality due to diarrhea in children in resource-poor settings (Qadri et al, 2005), (Bourgeois et al, 2016), (Liu et al, 2016), (Chakraborty et al, 2018)
Since there was no significant difference between the responses to labile toxins (LT) antigen with and without GM1 gangliosides, the multiplex assays were performed without GM1
The purpose of this study was to evaluate the feasibility of developing a multiplex enzyme linked immunosorbent assay (ELISA) for assessing the frequency, magnitude and breadth of serum and mucosal responses to ETEC colonization factors (CFs) and toxin antigens using the Meso Scale Discovery (MSD) platform

Summary

Introduction

Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of morbidity and mortality due to diarrhea in children in resource-poor settings (Qadri et al, 2005), (Bourgeois et al, 2016), (Liu et al, 2016), (Chakraborty et al, 2018). A significant road block to successful vaccine development is our poor understanding of the antigens that elicit protective immune responses against ETEC illness and the immune mechanisms of protection (Chakraborty et al, 2015), Qadri et al, 2005), (Zhang and Sack, 2012)

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Conclusion