Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model

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 (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) 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 (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) 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 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

Figure 1. Construction and detection of the 3xSTa_A14Q-tmLT toxoid fusion.

Panel A: Amino acid sequences of the native STa toxin and the STa_A14Q toxoid. Panel B: Construction of the 3xSTa_A14Q-tmLT toxoid fusion gene with 3 copies of the STa_A14Q toxoid gene genetically fused at the 5’ end, within LT_A, and the 3’ end of the tmLT toxoid gene (LT_{S63K/R192G/L211A}). The numbers and arrows indicated primers used in PCRs to mutate the genes and to amplify fragments to be overlapped for a single open reading frame encoding the 3xSTa_A14Q-tmLT toxoid fusion. This chimeric toxoid fusion gene was cloned in vector pET28α and expressed in E. coli BL21 as a 6xHis-tagged fusion protein. The drawing scale is not in proportion to nucleotide fragment sizes. Panel C: Detection of 6xHis-tagged 3xSTa_A14Q-tmLT toxoid fusion protein in Western blot using 12% PAGE gel, with rabbit anti-CT antiserum (1:3300; Sigma) and IRDye-labeled goat anti-rabbit IgG (1:5000; LI-COR, Lincoln, NE). Panel D: Detection of the toxoid fusion protein with purified rabbit anti-STa antiserum (1:5000) and IRDye-labeled goat anti-rabbit IgG (1:5000; LI-COR, Lincoln, NE). Extracted 6xHis-tagged protein form fusion strain 9164 and total protein extracts from negative control strain 8955 were examined in the SDS-PAGE. Lane M is the protein marker (Precision Plus Protein Pre-stained standards, Bio-Rad, Hercules, CA).

Figure 2. Fusion protein toxicity assays in T-84 cells using cAMP and cGMP EIA ELISA kits.

200 ng or 2 µg of the refolded toxoid fusion protein 3xSTa_A14Q-tmLT were incubated with T-84 cells, and intracellular cAMP (panel A) and cGMP (panel B) levels (pmol/ml) were measured. T-84 cells incubated with the toxoid fusion stimulated significant less cAMP (p<0.01) or cGMP (p<0.01) compared to those incubated with 2 ng STa or 10 ng CT.

Figure 3. Anti-STa IgG and IgA antibody titration of serum and intestinal wash samples of IP or IN immunized mice (solid dots) and the control mice (circles).

Panel A: anti-STa IgG and IgA antibodies titers in the serum and intestine wash samples of the IP immunized mice, but not in the control mice (p<0.01). Panel B: anti-STa IgG and IgA antibodies titers from serum and intestine wash samples of the IN immunized mice, and no anti-STa IgG or IgA antibodies detected in the control mice (p<0.01). For STa antibody titration ELISAs, 1.3 - 2 ng STa-ovalbumin conjugates were used to coat each well of a Costar plate (Nunc); HRP-conjugated goat-anti-mouse IgG (1:3000) or IgA (1:1000) as the secondary antibodies. Optical densities of greater than 0.4 (after subtracting the background reading) were used to calculate anti-STa antibody titers (in log₁₀).

Figure 4. Anti-LT antibody titration from serum, fecal suspension, and intestine wash samples of the IP and IN immunized mice.

Panel A: anti-LT IgG and IgA antibody titers from serum and intestine wash samples of the IP immunized (solid dots) and control mice (circles) (p<0.01). Panel B: anti-LT IgG and IgA antibody titers from serum samples of the mice IN immunized with 130 µg fusion antigen with 2 µg CT adjuvant or 2 µg CT adjuvant alone, but not of mice immunized with 130 µg fusion antigen without CT or Tris-HCl buffer only. Panel C: anti-LT IgA antibody titers detected from fecal suspension samples of mice IN immunized with the fusion antigen with 2 µg CT adjuvant or CT adjuvant alone, but not of mice immunized with fusion antigen without CT or Tris-HCl buffer. Panel D: anti-LT IgG and IgA antibody titers detected in intestinal wash samples of the mice IN immunized with the fusion and CT or CT, but not with the fusion alone or Tri-HCl buffer. Anti-LT antibody titration ELISAs used 25 ng CT (Sigma) to coat each well of an Immulon 2HB plate (Thermo Scientific); HRP-conjugated goat-anti-mouse IgG (1:5000) or IgA (1:1000) as the secondary antibodies. Optical densities of greater than 0.4 (after subtracting the background reading) were used to calculate anti-LT antibody titers (in log₁₀).

Figure 5. Neutralization against CT from antibodies in pooled serum (1:33.3 dilution), fecal (1:200 dilution) and intestine washes (1:83.3 dilution) of the IP and IN immunized mice.

Panel A: serum, fecal suspension and intestine wash samples of the IP immunized mice (solid boxes) and the control mice (open boxes) were examined for neutralizing against 10 ng CT in T-84 cells using EIA cyclic AMP ELISA kit (cAMP EIA, Assay Design). Panel B: serum, fecal suspension and intestine wash samples of mice IN immunized with the 3xSTa_A14Q-tmLT toxoid fusion with CT (solid boxes) or CT adjuvant (open boxes) were examined for neutralization against 10 ng CT. Intracellular cAMP concentrations (pmol/ml) were measured by following the manufacture’s protocol. Boxes and error bars indicate means and standard deviations.

Figure 6. Neutralization against STa toxin from antibodies in pooled serum (1:33.3), fecal suspension (1:200) and intestine washes (1:83.3) of the IP and IN immunized mice.

Panel A: serum, fecal suspension and intestine wash samples of the IP immunized mice (solid boxes) and control mice (open boxes) were examined for neutralization against 2 ng STa in T-84 cells using EIA cyclic GMP ELISA kit (cGMP EIA, Assay Design). Panel B: serum, fecal suspension and intestine wash samples of mice IN immunized with the 3xSTa_A14Q-tmLT toxoid fusion antigen with CT (solid boxes) or CT adjuvant (open boxes) were examined for neutralization against 2 ng STa. Boxes and error bars indicate means and standard deviations.