Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTa _N12S -mnLT _G192G/L211A -Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea

Abstract

Enterotoxigenic (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTa-dmLT or CFA/I/II/IV-2xSTa-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STa, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa or LT ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTa-mnLT induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa ETEC and LT ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTa-mnLT induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

Impact of TGEV infection on the pig small intestine

“…Based on PCR analysis, we observed fewer Lactobacillus and more Enterobacteriaceae in TGEV-infected pigs. Among Enterobacteriaceae family, Escherichia coli is the most common cause of diarrhea in pigs and piglets [22][23][24]. Research has shown that animals with signs of intestinal disease have far greater numbers of Enterobacteriaceae than do healthy individuals, and that the Enterobacteriaceae are linked to intestinal disorders [25,26].…”

Adjuvant effect of enterotoxigenic Escherichia coli (ETEC) double-mutant heat-labile toxin (dmLT) on systemic immunogenicity induced by the CFA/I/II/IV MEFA ETEC vaccine: Dose-related enhancement of antibody responses to seven ETEC adhesins (CFA/I, CS1-CS6)

“…Mouse serum anti-CFA adhesin and anti-LT IgG antibody titers were measured in ELISAs as previously described. [23][24][25] Briefly, wells of 2HB plates (Thermo Scientific, Rochester, NY) coated with 100 ng of heat-extracted fimbriae (CFA/I, CS1, CS2, CS3, CS4, CS5), recombinant CS6 subunit protein CssA, or cholera toxin (CT; Sigma, St. Louis, MO) were incubated with two-fold diluted mouse serum samples (from 1:200 to 1:51200) for 1h at 37°C. Horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:3000; Bethyl Laboratories, Montgomery, TX) and 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD) were used to measure optical density (OD 650 ).…”

“…[14][15][16][17][18][19][20][21] Recent studies suggested that dmLT adjuvant might have positive immunoregulatory properties when given parenterally with vaccine antigens. 22,23 The potential impact of adding dmLT to a parenteral subunit ETEC vaccine, however, has not been fully investigated. In this study, we subcutaneously (SC) immunized mice with ETEC adhesin MEFA (multiepitope fusion antigen) CFA/I/II/IV, a leading and novel subunit ETEC vaccine candidate, and examined dmLT adjuvant activity in up-regulating antibody responses to the seven ETEC adhesin antigens included in the vaccine construct, CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6).…”

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

“…By mimicking epitope native antigenicity and presenting multiple heterologous neutralizing epitopes at a backbone immunogen, MEFA vaccinology allows a single immunogen (protein) to carry multiple antigenic elements (epitopes or peptides) from various virulence determinants for broad immunogenicity, thus allowing the development of a broadly immunogenic multivalent vaccine. With this MEFA technology, we have successfully generated ETEC fimbria MEFAs for broadly protective immunity against seven or nine human ETEC virulence determinants (colonization factor antigen [CFA] adhesins) (9-11) and adhesin-toxoid MEFAs for antibodies not only inhibiting adherence of seven human ETEC adhesins and neutralizing LT and STa enterotoxicity but also protecting against ETEC diarrhea in a pig challenge model (12,13). These MEFAs have become leading antigens for human ETEC subunit vaccines against children's diarrhea and traveler's diarrhea.…”