Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7

Abstract

Infection with Escherichia coli O157:H7 may develop into hemorrhagic colitis, or hemolytic uremic syndrome (HUS), which usually causes kidney failure or even death. The adhesion and toxins are the important virulent factors. In this study, a novel vaccine candidate rSOBGs was constructed based on the bacterial ghost (BG). rSOBGs maintained the integrity of cellular morphology and displayed the linear Stx2Am-Stx1B antigen on the surface of outer membrane. rSOBGs induced Stxs-specific IgA/IgG antibodies and stronger intimin-specific IgA/IgG antibodies effectively in sera in this study. In vivo, the rSOBGs provided the higher protection rate (52%) than native bacterial ghost-OBGs (12%) when challenged intragastricly with high dose (500 LD50) viable E. coli O157:H7. Meanwhile, the rSOBGs provided higher protection rate (73.33%) than OBGs when challenged with 2 LD50 even to 5 LD50 lysed E. coli O157:H7. In vitro, the rSOBGs-immunized sera possessed neutralizing activity to lysed pathogenic bacteria. Furthermore, the results of histopathology also displayed that the administration of rSOBGs have the ability to reduce or inhibit the adhesion lesions and toxins damages of organs. The novel vaccine candidate rSOBGs induced both anti-toxin and anti-adhesion immune protection, suggesting the possibility to prevent the infectious diseases caused by Escherichia coli O157:H7.

Figure 1. Verification of the surface antigens and evaluation of the cytotoxicity.

(A). Verification of the surface antigens. Flow cytometry was adopted to detect 3 × 10⁴ Hep-2 cells. BGs were used as antigens, PBS contains chicken anti-Stx1B IgY, rabbit anti-Stx2A sera or rabbit anti-intimin sera was used as first anitibody, respectively. HRP labeled anti-chicken IgG or anti-rabbit IgG was used as test antibody according to their sources. (B). Cytotoxicity analysis of rSOBGs. Serial diluted BGs were incubated with Vero cell monolayer for 36 h, and the MTT assay was performed to detect the cytotoxicity. *P < 0.01 vs PBS group.

Figure 2. Detection of the specific IgA and IgG titers in sera and irrigating solution of immunized mice.

ELISA was used to detect the levels of specific antibodies. OBGs, intimin, Stx1 or Stx2 was coated as antigen, respectively. Serial diluted serum or irrigating solution in PBS were added and incubated. HRP labeled anti-mouse IgA or IgG was used as the test antibody, respectively. (A). BGs-specific IgA and IgG antibodies in sera and irrigating solution. (B). intimin-specific IgA and IgG antibodies in sera and irrigating solution. (C). Stx1-specific IgA and IgG antibodies in sera and irrigating solution. (D). Stx2-specific IgA and IgG antibodies in sera and irrigating solution. *P < 0.01 vs PBS group; **P < 0.01; ^#P > 0.05 vs PBS group; ^##P > 0.05.

Figure 3. Protection of rSOBGs-immunized mice against lethal dose challenge of viable and lysed O157:H7.

The male BALB/c mice immunized on day 0 for primary immunization, and immunized on day 14 for boost. 14 days post the last immunization, the mice were challenged. (A). Mice were intragastric challenged with 10⁹ CFU viable O157:H7 EDL933. (B). Mice were intragastric challenged with 5 × 10⁹ CFU viable O157:H7 EDL933. (C). Mice were challenged i.p. with 2 × 10⁸ CFU lysed O157:H7 88321. (D). Mice were challenged i.p. with 5 × 10⁸ CFU lysed O157:H7 88321. *P < 0.01 vs PBS group; **P < 0.01; ^##P > 0.05.

Figure 4. Pathology of liver, kidney and intestine of rOBGs immunized mice.

Sections of paraffin-embedded tissue were stained with hematoxylin and eosin, and examined under light microscopy. Pathological changes were labeled by arrows. (A). Normal mice. (B). Mice succumbing to intragastric challenge with viable O157:H7 EDL933. (C). Survival mice intragastric challenged with viable O157:H7 EDL933. (D). Mice succumbing to challenge with lysed O157:H7 88321. E. Survival mice challenged i.p. with lysed O157:H7 88321.

Figure 5. In vitro protection of antisera against lysed O157:H7.

The antisera were incubated with different lethal dose lysed O157:H7 88321, then these samples were challenged i.p. in BALB/c mice. The survival was observed for 14 days. (A). The neutralizing capacity against 2 × 10⁸ lysed O157:H7 88321 in vitro. (B). The neutralizing capacity against 5 × 10⁸ lysed O157:H7 88321 in vitro. **P < 0.01 vs PBS group.