Antibodies induced by enterotoxigenic Escherichia coli (ETEC) adhesin major structural subunit and minor tip adhesin subunit equivalently inhibit bacteria adherence in vitro.

Hyesuk Seo,Zhenhai Chen,Weiping Zhang,Qiangde Duan,Jerome Nietfeld,Rahul M Nandre,Nicholas J Mantis

doi:10.1371/journal.pone.0216076

Hyesuk Seo, Zhenhai Chen + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0216076

Copy DOI

Abstract

Antibodies that block the adherence of enterotoxigenic Escherichia coli (ETEC) to host intestinal epithelial cells are protective. Multiepitope-fusion-antigens (MEFAs) carrying epitopes of ETEC adhesin major subunits or tip minor subunits induced antibodies against ETEC adherence. Adherence inhibition effectiveness of antibodies induced by major subunit epitopes versus minor tip subunit epitopes, however, has not been comparatively characterized. In this study, we immunized mice with a major subunit MEFA or a tip MEFA, evaluated MEFA anti-adhesin immunogenicity, and examined induced-antibodies against bacteria in vitro adherence or in vivo colonization in mice. Mice subcutaneously immunized with major subunit MEFA CFA/I/II/IV or tip MEFA showed no adverse effects and developed strong antigen-specific antibody responses. Data showed that antibodies derived from two MEFAs were equally effective against adherence of the bacteria expressing CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin in vitro. Subsequently, we immunized mice with CFA/I fimbriae, major subunit CfaB, or minor tip adhesin subunit CfaE. We found that antibodies induced by CFA/I, CfaB and CfaE equally inhibited in vitro adherence of ETEC strain H10407. Furthermore, we immunized mice with CFA/I fimbriae, CfaB, or CfaE, and then challenged the mice with H10407. Data showed that although not significantly, fewer H10407 bacteria colonized the immunized mice. These results suggest that ETEC adhesin major subunit and minor tip subunit should be equally effective in inducing neutralizing anti-adhesin antibodies, and that major subunit CFA/I/II/IV MEFA or tip MEFA, perhaps combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A, can be used for development of broadly protective vaccines against ETEC diarrhea.

Highlights

Diarrheal disease remains a major global health problem as it continues to be a leading cause of death in children younger than 5 years of age in the developing countries [1, 2]
Mice SC immunized with major subunit CFA/I/II/IV MEFA alone or the major subunit MEFA premixed with toxoid fusion 3xSTaN12S-mnLTR192G/L211A developed anti-adhesin IgG antibody responses (Fig 1)
Mouse serum anti-CFA/I, -CS1, -CS2, -CS3, -CS4, -CS5, and antiCS6 IgG titers were detected at 3.1 ± 0.23, 3.1 ± 0.20, 2.3 ± 0.21, 3.4 ± 0.13, 3.2 ± 0.23, 3.5 ± 0.15 and 3.7 ± 0.25 in the group immunized with major subunit CFA/I/II/IV MEFA alone, and 3.8 ± 0.21, 3.3 ± 0.15, 2.4 ± 0.24, 3.5 ± 0.16, 3.5 ± 0.14, 3.6 ± 0.14, and 3.8 ± 0.22 in the group co-immunized with the major subunit MEFA and the toxoid fusion

Summary

Introduction

Diarrheal disease remains a major global health problem as it continues to be a leading cause of death in children younger than 5 years of age in the developing countries [1, 2]. We recently developed a structure- and epitope-based vaccine technology called MEFA, multiepitope fusion antigen By applying this MEFA technology, we constructed multivalent MEFA immunogens by embeding epitopes from multiple fimbrial major subunits or minor tip adhesin subunits in a backbone ETEC subunit protein (via epitope substitution); we subsequently explored the potential of MEFAs in developing broadly protective anti-adhesin subunit vaccines against ETEC diarrhea. Efficacy against bacterial adherence from antibodies induced by major subunit CFA/I/II/IV MEFA and tip MEFA has never been comparatively examined It has never been examined whether antibodies to a fimbrial subunit (major or minor)and antibodies to an entire fimbria can block ETEC bacterial adherence. We immunized mice with entire CFA/I fimbria, CFA/I fimbrial major structural subunit CfaB, or fimbrial tip adhesin subunit CfaE to assess induced antibodies for efficacy against ETEC bacterial adherence. We further explored the potential application of CFA/I/II/IV MEFA or tip MEFA, co-administered with toxoid fusion 3xSTaN12S-mnLTR192G/L211A (previously labeled as 3xSTaN12S-dmLT) which has been demonstrated to induce antibodies protecting against ETEC heat-labile toxin (LT) and heat-stable toxin (STa) [18], for vaccine development against ETEC diarrhea

Methods and materials

Results

Discussion