Significance of Enterotoxigenic Escherichia coli (ETEC) Heat-Labile Toxin (LT) Enzymatic Subunit Epitopes in LT Enterotoxicity and Immunogenicity

Abstract

Enterotoxigenic (ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM-binding LT is a strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LT subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes from LT subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LT subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LT epitopes were immunogenic; epitope 7 (SPHPYEQEVSA) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LT subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM-binding activity. The results from this study indicated that while not all immunodominant LT epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases. No vaccine is licensed for enterotoxigenic (ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC)

“…PyMOL Molecular Graphics System (version 2.2; Schrödinger, LLC, New York City, NY, USA) was used to display the location of each epitope on the FedF protein. Nucleotides coding each FedF epitope were embedded into CfaB gene by replacing nucleotides coding 80-86 amino acids of CfaB (a CfaB epitope) using SOE PCR with specifically designed primers (Table 2), as we previously described Huang et al, 2018;Lu and Zhang, 2017). PCR generated CfaB-epitope fusion genes were digested with NheI and EagI restriction enzymes (New England BioLabs, Ipswich, MA), and were cloned into pET28a vector.…”

“…Recombinant CfaB-epitope (CfaB-FedF-ep) fusion proteins expressed by E. coli BL21 (DE3) were extracted with bacterial protein extraction reagent (B-PER; Thermo Fisher Scientific, Rochester, NY) and refolded as we previously described (Huang et al, 2018;Nandre et al, 2016;Rausch et al, 2017). Refolded fusion proteins were examined in SDS-PAGE with Coomassie blue staining, and were characterized in Western blot or direct ELISAs with anti-F18 mouse antiserum.…”

“…Neutralizing epitopes from K88 fimbrial adhesin FaeG (Lu and Zhang, 2017) and toxins including LT (Huang et al, 2018), STa (Rausch et al, 2017;Ruan et al, 2014b;Zhang et al, 2010), and STb (Rausch et al, 2017) were identified. However, epitopes from F18 adhesive subunit FedF are not mapped and neutralizing epitopes have not been identified.…”

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG

“…Protective epitopes of four toxins (LT, STa, STb, and Stx) produced by ETEC strains associated with pig diarrhea were identified (14)(15)(16)(17). MEFA technology has also assisted with the construction of a multivalent toxin antigen and the development of a broadly protective antitoxin vaccine candidate for pig postweaning diarrhea (15).…”

A bivalent vaccine confers immunogenicity and protection against Shigella flexneri and enterotoxigenic Escherichia coli infections in mice

“…Similarly, CFA/I is one of the most common ETEC colonization factors and has been used as a target for ETEC vaccine 11 . LTb is also a common antigen used in the vaccine studies 12 . Overall, the current findings expand our knowledge about the immunogenic potential of these antigens for further vaccine studies.…”