Passive antibodies derived from intramuscularly immunized toxoid fusion 3xSTa N12S -dmLT protect against STa+ enterotoxigenic Escherichia coli (ETEC) diarrhea in a pig model

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are among the most common causes of children’s diarrhea and travelers’ diarrhea. Developing effective vaccines against ETEC associated diarrhea becomes a top priority. ETEC heat-labile toxin (LT) and heat-stable toxin (STa) toxoid fusion 3xSTaN12S-dmLT was demonstrated recently to induce neutralizing antitoxin antibodies in intraperitoneally or subcutaneously immunized mice. However, whether antibodies derived from this toxoid fusion are protective against ETEC diarrhea has not been examined. In this study, we intramuscularly immunized pregnant gilts with toxoid fusion 3xSTaN12S-dmLT, challenged suckling piglets with a STa-positive ETEC strain, and assessed protective efficacy of passive acquire antitoxin antibodies against ETEC diarrhea. Data showed all three immunized gilts developed anti-STa IgG and IgA antibodies, and piglets born to the immunized dams acquired anti-STa and anti-LT antibodies. When challenged with a STa+ ETEC strain, none of the piglets born to the immunized dams developed watery diarrhea, with 20 piglets remained normal and the other 8 piglets developed mild diarrhea indicated with stained butt. In contrast, the control dams and born piglets had no anti-STa or anti-LT antibodies detected, and 26 out 32 piglets developed watery diarrhea after challenge of the STa+ ETEC strain. These results indicated that passive acquired anti-STa antibodies are protective against ETEC diarrhea, and suggested potential application of toxoid fusion 3xSTaN12S-dmLT in ETEC vaccine development.

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].…”

Development of an enterotoxigenic Escherichia coli vaccine based on the heat-stable toxin

“…57,81,[89][90][91][92] Three copies of an STh-N12S mutant genetically fused with dmLT, 3xSTh N12S -dmLT, was identified as a promising vaccine candidate that may engender neutralizing antibodies against both ST and LT. 84,85,92 Notably, piglets born from 3xSTh N12S -dmLT immunized pregnant gilts seemed to be passively protected from ETEC diarrhea during challenge. 85 The 3xSTh N12S -dmLT construct was further genetically fused to seven putative immunodominant colonization factor epitopes from prevalent ETEC strains (CFA/I/II/IV). 57 Interestingly, the expressed single polypeptide immunogen elicited anti-adhesin antibodies against all the seven CFA in addition to anti-ST and anti-LT antibodies in mice.…”

“…57,58 The non-toxic double mutant LT (dmLT; LT R192G/L211A ) has also been used as a carrier, offering the additional benefit of engendering neutralizing anti-LT antibodies. [83][84][85] Additionally, an LT-STh P13F fusion was incorporated into a live attenuated oral E. coli vaccine (ZCR533) that constitutively express the antigen on its surface or as a secretion. 86…”

“…92 The STh-N12S seems to be the most promising one, and is the current lead candidate mutation for the LT-based genetic fusions reviewed above. 84,85 In our mutational study of all possible 361 single-amino acid substitutions of STh, where we screened for effects on toxicity and antigenicity, the top 30 toxoid candidates (based on their antigenicity-toxicity ratios) had mutations either in L9, N12 or A14. 45 Notably, 17 A14 mutation toxoids had undetectable toxicities in the T84 cell assay, 45 suggesting that A14 is the prime residue to target for abolishing toxicity.…”

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

“…Recently, ETEC adhesin antigen CFA/I/II/IV MEFA (multiepitope fusion antigen), carrying epitopes of the seven most prevalent adhesins (CFA/I, CFA/II [CS1, CS2, and CS3], and CFA/IV [CS4, CS5, and CS6]), was demonstrated to induce antibodies against adherence of ETEC and E. coli bacteria expressing these seven CFA adhesins in vitro (18). Recent studies also showed that genetic fusions of an STa toxoid and a monomeric LT (mnLT; one LT A domain and one LT B domain as a single peptide) induced antitoxin antibodies to neutralize both toxins (19)(20)(21)(22)(23)(24).…”