Toxicity and Immunogenicity of Enterotoxigenic Escherichia coli Heat-Labile and Heat-Stable Toxoid Fusion 3xSTaA14Q-LTS63K/R192G/L211A in a Murine Model

Chengxian Zhang,Weiping Zhang,David E Knudsen,Mei Liu,Donald C Robertson,Mrutyunjay Suar

doi:10.1371/journal.pone.0077386

Abstract

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STaP13F) fused at the N- or C-terminus, or inside the A subunit of LTR192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STaA14Q) and a triple-mutant LT toxoid (LTS63K/R192G/L211A, tmLT), constructed a toxoid fusion (3xSTaA14Q-tmLT) that carried 3 copies of STaA14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTaA14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

Highlights

Diarrhea is the second leading cause of death to young children who live in developing countries [1], and continues to be a major threat to global health [2]
Cloning of the mutated stable toxin type Ib (STa) or labile toxin (LT) genes in each mutant strain was verified from DNA sequencing, and expression and secretion of the STa and LT proteins were confirmed from STa competitive ELISA and GM1 ELISA, respectively
Cyclic GMP ELISA showed the cGMP level in T-84 cells incubated with culture supernatant of STaA14Q mutant strain 8407 (Table 1) was 0.093 pmole/ml, a level significantly lower (p=0.007, p

Summary

Introduction

Diarrhea is the second leading cause of death to young children who live in developing countries [1], and continues to be a major threat to global health [2]. ETEC strains are the most common cause of diarrhea to children and adults travelling to ETEC endemic countries or regions, military personnel deployed at these areas, and immunocompromised patients [2,4,5,6]. These ETEC strains produce various bacterial adhesins and one or more enterotoxins. There are no effective vaccines currently available to protect against ETEC diarrhea [10], despite the facts that the association between E. coli and children diarrhea was discovered over 100 years ago [11], that the disease mechanism of ETECassociated diarrhea has been well studied [8,10], and that ETEC strains have been identified the leading bacteria that cause diarrhea [2]

Methods

Results

Conclusion