Co-administered Tag-Less Toxoid Fusion 3xSTaN12S-mnLTR192G/L211A and CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) Induce Neutralizing Antibodies to 7 Adhesins (CFA/I, CS1-CS6) and Both Enterotoxins (LT, STa) of Enterotoxigenic Escherichia coli (ETEC)

Abstract

Enterotoxigenic Escherichia coli (ETEC) bacteria remain a leading cause of children's diarrhea and travelers' diarrhea. Vaccines that induce antibodies to block ETEC bacterial adherence and to neutralize toxin enterotoxicity can be effective against ETEC-associated diarrhea. Recent studies showed that 6xHis-tagged CFA/I/II/IV multiepitope fusion antigen (MEFA) induced broad-spectrum antibodies to inhibit adherence of the seven most important ETEC adhesins (CFA/I, CS1 to CS6) (Ruan et al., 2014a) and 6xHis-tagged toxoid fusion antigen 3xSTa_N12S-mnLT_R192G/L211A (previously named as 3xSTa_N12S-dmLT) elicited antibodies to neutralize both heat-labile toxin (LT) and heat-stable toxin (STa) produced by ETEC strains (Ruan et al., 2014b). In this study, we constructed two new genes to express tag-less toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A and tag-less CFA/I/II/IV MEFA and then examined immunogenicity of each tag-less protein in mouse immunization. We further combined two tag-less proteins and investigated antigen co-administration in mice. Data showed that mice immunized with tag-less 3xSTa_N12S-mnLT_R192G/L211A or tag-less CFA/I/II/IV MEFA developed antigen-specific IgG antibody responses, and mice co-administered with two tag-less proteins induced neutralizing antibodies against seven adhesins and both toxins. These results indicated tag-less toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A and tag-less CFA/I/II/IV MEFA administered individually or combined induced neutralizing antitoxin and/or anti-adhesin antibodies, and suggested the potential application of two tag-less proteins for ETEC vaccine development.

Keywords: ETEC (enterotoxigenic Escherichia coli); MEFA (multiepitope fusion antigen); antibody; diarrhea; toxoid fusion; vaccine.

Figure 1

Tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) recombinant protein expression, characterization and immunogenicity. (A) Coomassie blue staining to show refolded tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) and 6xHis-tagged 3xSTa_N12S-mnLT_R192G/L211A (9331) proteins electrophoresed in 12% SDS-PAGE. (B) Western blot detection of tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) and 6xHis-tagged 3xSTa_N12S-mnLT_R192G/L211A (9331) proteins with anti-CT rabbit antiserum. (C) Western blot detection of tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) and 6xHis-tagged 3xSTa_N12S-mnLT_R192G/L211A (9331) with anti-STa rabbit antiserum. (D) anti-STa and anti-LT IgG antibody titers (log₁₀) detected from the serum samples of the mice IP immunized with tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) with dmLT adjuvant or the control mice (without injection); six mice per group. Boxes and bars indicate means and standard deviations. Molecular weight marker in kDa.

Figure 2

Tag-less CFA/I/II/IV MEFA (9472) protein expression, characterization, and immunogenicity. (A) SDS-PAGE Coomassie blue staining showed the refolded 6xHis-tagged CFA/I/II/IV MEFA (9175; ~17 kDa) and tag-less CFA/I/II/IV MEFA proteins (9472; ~15 kDa). (B) Western blot detection of the refolded tag-less CFA/I/II/IV MEFA (9472), 6xHis-tagged (9175) proteins, or total proteins of E. coli BL21 host strain, with mouse anti-CFA/I antiserum. (C) IgG antibody titers (in log₁₀) from the serum samples of the mice IP immunized with the tag-less CFA/I/II/IV MEFA (9472; open box) or the control group (solid box; without injection); dmLT was used as the adjuvant; six mice per group. Boxes and bars indicate means and standard deviations (log₁₀). Molecular weight marker in kDa.

Figure 3

Protein thermal stability assessment of refolded tag-less CFA/I/II/IV MEFA (9472). Tag-less CFA/I/II/IV MEFA protein was characterized by SDS PAGE Coomassie blue staining (top) and Western blot using anti-CFA/I antiserum (bottom), after exposure at 37 or 50°C for 1, 2, 5, 7, 10, 14, 18, 21, 28, 35, or 42 days. M, molecular weight marker (kDa).

Figure 4

Mouse serum antibody in vitro neutralization activities against STa and CT enterotoxicity, measured with T-84 cells and a cGMP or a cAMP EIA kit (Enzo Life Sciences). (A) The serum samples of the mice IP immunized with tag-less toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A (9471) or 6xHis-tagged 3xSTa_N12S-mnLT_R192G/L211A (9331) prevented STa toxin from elevating intracellular cyclic GMP in T-84 cells, whereas serum from the control mice (without injection) did not prevent STa from stimulating cGMP in T-84 cells. (B) The serum samples from the mice IP immunized with tag-less 3xSTa_N12S-mnLT_R192G/L211A (9471) or 6xHis-tagged 3xSTa_N12S-mnLT_R192G/L211A (9331) prevented CT toxin from elevating intracellular cyclic AMP in T-84 cells.

Figure 5

IgG antibody titers (log₁₀) from the serum samples of the mice IP co-immunized with tag-less toxoid fusion 3xSTa_N12S-mnLT_R192G/L211A (9471) and tag-less CFA/I/II/IV MEFA (9472), or the control mice (without injection). Six mice per group. Boxes and bars indicate means and standard deviations (log₁₀).