A multivalent vaccine candidate targeting enterotoxigenic Escherichia coli fimbriae for broadly protecting against porcine post-weaning diarrhea

Qiangde Duan,Xiaojun Wang,Siguo Liu,Weiping Zhang,Boyu Jiang,Wenwen Wu,Guoqiang Zhu,Shengmei Pang,Zhiming Pan

doi:10.1186/s13567-020-00818-5

Abstract

Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG–FedF–FanC–FasA–Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG–FedF–FanC–FasA–Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

Highlights

Enterotoxigenic Escherichia coli (ETEC) characteristically produces two types of virulence factors: fimbriae and enterotoxins
Fimbriae‐targeted multi-epitope fusion antigen (MEFA) carried the epitopes of the five fimbriae subunits of porcine ETEC In silico predictions for each fimbriae’s antigens indicate two epitopes (‘NVGNGSGGANIN’ and ‘QLKKDDRAPSNGGYK’) from the K99 fimbria major subunit FanC, two epitopes (‘LAAPAENNTSQAN’ and ‘AGNNNTGSDTKYLV PASNDTSASG’) from the 987P fimbria major subunit FasA, and two epitopes (‘VMAADWTEGQ PGDII’ and ‘WDDLSHPNYTSADKASYLSYGSGVSAG’) from the F41 fimbria major subunit Fim41a
The faeg–fim41a–fanc– fasa and fedf–fasa–fim41a–fanc genes were fused to a faeg–fedf–fanc–fasa–fim41a fimbriae MEFA gene using the splicing overlap extension (SOE)-splicing overlap extension PCR (PCR) method (Figure 1A)

Summary

Introduction

Enterotoxigenic Escherichia coli (ETEC) characteristically produces two types of virulence factors: fimbriae and enterotoxins. Despite the difference in the antigenic classification of K88 serological variants (e.g., K88ab, ac, ad), the major structural subunit—FaeG—has been recognized as the common adhesive subunit to all variants [8]. When it comes to the F18 fimbria, two antigenic variants—F18ab and F18ac—have been identified. Once colonization of the small intestinal epithelial cells occurs, ETEC bacteria secrete two classes of enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). These toxins stimulate the intestinal lining to secrete excessive fluid, causing diarrhea. The initial subsequent attachment between the adhesive subunit of the fimbriae and special receptors on host cells facilitates efficient delivery of enterotoxins and progression of infection pathogenesis, both of which play important roles in ETEC infections

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