A Novel Vaccine for Bovine Diarrhea Complex Utilizing Recombinant Enterotoxigenic Escherichia coli and Salmonella Expressing Surface-Displayed Chimeric Antigens from Enterohemorrhagic Escherichia coli O157:H7

Abstract

Background/objectives: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a zoonotic pathogen primarily found in cattle, causes Hemolytic Uremic Syndrome (HUS) in humans, often through contaminated food. Its Type Three Secretion System (T3SS) facilitates gut colonization. In contrast, neonatal calf diarrhea (NCD) is mainly caused by pathogens like enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Bovine Coronavirus (BCoV), and Bovine Rotavirus type A (BRoVA). This study engineered a chimeric protein combining EspB and Int280γ, two T3SS components, expressed in the membranes of Salmonella Dublin and ETEC.

Methods: Immune responses in vaccinated mice and guinea pigs were assessed through ELISA assays.

Results: Successful membrane anchorage and stability of the chimera were confirmed. Immune evaluations showed no enhancement from combining recombinant bacteria, indicating either bacterium suffices in a single formulation. Chimeric expression yielded immunogenicity equivalent to 10 µg of recombinant protein, with similar antibody titers. IgG1/IgG2a levels and Th1, Th2, and Th17 markers indicated a mixed immune response, providing broad humoral and cellular protection. Responses to BCoV, BRoVA, ETEC, and Salmonella antigens remained strong and did not interfere with chimera-specific responses, potentially boosting NCD vaccine efficacy.

Conclusions: The chimera demonstrated robust immunogenicity, supporting its potential as a viable vaccine candidate against EHEC O157:H7. This approach could enhance NCD vaccine valency by offering broader protection against calf diarrhea while reducing HUS transmission risks to humans.

Keywords: EHEC O157:H7; ETEC; EspB; Salmonella; bovine vaccine; coronavirus; immune response; intimin; rotavirus.

Figure 1

(A) Schematic representation of the chimeric protein. The chimera comprises a Wza-Omporf1 anchor sequence that inserts itself into the outer membrane (OM) at the N-terminal end and orients the rest of the EspB-linker-Int280 antigenic fusion to the extracellular milieu. The C-terminal region ends with the epitopes for c-Myc and His6x-tags. The approximate molecular weight of the chimera is 74 kDa. Detection of the recombinant chimeric protein expression and outer membrane localization in recombinant ETEC and Salmonella. (B) ETEC and Salmonella transformed with pTrcHis2B-BLI280 were induced (I), or not (non-induced: NI), with IPTG for recombinant chimera expression. The samples were separated by SDS-PAGE. Wild-type ETEC and Salmonella strains were used as controls not expressing the chimera and recombinant Int280ϒ-Hisx6 (RP) purified protein was used as primary antibody control. (C) ETEC and Salmonella transformed with pTrcHis2B-BLI280 were induced with IPTG to express the recombinant chimera. Samples of different fractions from the membrane purification process of ETEC and Salmonella were separated by SDS-PAGE. References: outer membrane (OM), pellet washings (W), pellet supernatant (SN), total bacterial pellet (P) and total bacterial culture supernatant (C). Chimera detection was performed using a mouse-specific anti Hisx6-tag primary antibody and an alkaline phosphatase-conjugated anti-mouse as a secondary antibody, in both Western blot assays.

Figure 2

Detection of recombinant chimeric protein in inactivated recombinant ETEC and Salmonella. Induced recombinant ETEC and Salmonella were inactivated with 0.2% formalin incubated for 72 h at 4 °C. The samples were separated by SDS-PAGE. Chimera detection was performed using a mouse-specific anti Hisx6-tag primary antibody and an alkaline phosphatase-conjugated anti-mouse as a secondary antibody. The abbreviations correspond to uninduced bacteria (NI), induced bacteria (I) and induced and inactivated bacteria (II).

Figure 3

Scheme of immunization assays of mice and guinea pigs. Groups of mice and guinea pigs were immunized with two doses of vaccine preparations in an interval of 21 days via subcutaneous injection. Sera samples were collected from both animal models at days 1, 21 and 39. On day 39, the mice were sacrificed, and splenectomy was performed. An additional sera sample collection was performed for the guinea pigs on the 70th day before performing euthanasia of the animals. The mice were divided into seven groups of five animals each; one group of four animals was used as a control. The different groups were numbered from 1 to 8 and inoculated with the following antigens combinations: Group 1: PBS Control, Group 2: 1 µg of EspB and 1 µg of Int280ϒ, Group 3: 2 µg of the chimera, Group 4: 10 µg of the chimera, Group 5: ETEC expressing the chimera, Group 6: Salmonella expressing the chimera, Group 7: Both recombinant bacteria expressing the chimera and Group 8: Both recombinant bacteria expressing the chimera plus BCoV and BRoVA viral particles. The guinea pigs were divided into two groups of five animals each. One group was inoculated with a vaccine containing both recombinant bacteria expressing the chimera plus BCoV and BRoVA viral particles. The other group was vaccinated with PBS (Control).

Figure 4

Specific IgG responses at 39 dpv in inoculated mice. ELISA plates were coated with purified the recombinant chimera (A), EspB (B) and Int280ϒ (C), respectively. Specific antibodies response to the chimera, Int280ϒ and EspB were measured for each group using indirect ELISA and sera samples (39 dpv) from mice. Group 1: 150 µL of PBS (Control), Group 2: 1 µg of EspB and 1 µg of Int280γ, Group 3: 2 µg of the chimera, Group 4: 10 µg of the chimera, Group 5: 1.10⁸ inactivated CFU of ETEC B41Arg expressing the chimera, Group 6: 1.10⁸ inactivated CFU of Salmonella Dublin expressing the chimera, Group 7: 1.10⁸ inactivated CFU of ETEC B41Arg and Salmonella Dublin, both expressing the chimera and Group 8: 1.10⁸ inactivated CFU of ETEC B41Arg and Salmonella Dublin, both expressing the chimera, plus 1.10⁷ FFU BRoVA UK and BCoVB Mebus. Goat anti-mouse IgG conjugated with horseradish peroxidase was used as a secondary antibody. ABTS was used as substrate and the reaction was measured at OD₄₀₅. The antibody titer was expressed as the reciprocal of the end-point dilution resulting in an OD₄₀₅ above the cut-off value. The cut-off value was calculated as the average plus two times the standard deviation of the optical densities of the samples measured on day 0. Statistical analysis by ANOVA, p < 0.05 (*), p < 0.01 (**) and p < 0.0001 (****).

Figure 5

Specific IgG responses in inoculated guinea pigs. ELISA plates were coated with the purified recombinant chimera (A), EspB (B) and Int280ϒ (C), respectively. Specific antibodies response to the chimera, Int280ϒ and EspB were measured for each group using indirect ELISA and sera samples from guinea pigs. Goat anti-guinea pig IgG conjugated with horseradish peroxidase was used as a secondary antibody. ABTS was used as substrate and the reaction was measured at OD₄₀₅. The antibody titer was expressed as the reciprocal of the end-point dilution resulting in an OD₄₀₅ above the cut-off value. The cut-off value was calculated as the average plus two times the standard deviation of the optical densities of the samples measured on day 0. Statistical analysis by ANOVA p < 0.0002 (***) and p < 0.0001 (****).

Figure 6

IgG response against fimbria in sera of mice and guinea pigs inoculated with ETEC. ELISA plates were coated with purified fimbriae. Antibody response to fimbriae was measured for each group using indirect ELISA and sera samples from mice (A) and guinea pigs (B). Goat anti-guinea pig IgG conjugated with horseradish peroxidase was used as a secondary antibody. ABTS was used as a substrate, and the reaction was measured at OD₄₀₅. The antibody titer was expressed as the reciprocal of the end-point dilution resulting in an OD₄₀₅ above the cut-off value. The cut-off value was calculated as the average plus two times the standard deviation of the optical densities of the samples measured on day 0. Statistical analysis by ANOVA, p < 0.01 (**) and p < 0.001 (***).

Figure 7

IgG response against LPS in sera of inoculated mice and guinea pigs with Salmonella. ELISA plates were coated with purified LPS. Antibodies response to LPS were measured for each group using indirect ELISA and sera samples from of mice (A) and guinea pigs (B). Goat anti-guinea pig IgG conjugated with horseradish peroxidase was used as a secondary antibody. ABTS was used as a substrate and the reaction was measured at OD₄₀₅. The antibody titer was expressed as the reciprocal of the end-point dilution resulting in an OD₄₀₅ above the cut-off value. The cut-off value was calculated as the average plus two times the standard deviation of the optical densities of the samples measured on day 0. Statistical analysis by ANOVA, p < 0.05 (*) and p < 0.01 (**).

Figure 8

IgG response against BCoV and neutralizing antibodies against BRoVA in sera of vaccinated guinea pigs. (A) ELISA plates were coated with hyperimmune anti-BCoV serum. Clarified supernatants from HRT-18 cultures infected with standardized titer of coronavirus or supernatants from uninfected cells (control) was added into the corresponding wells. Commercial polyclonal anti-mouse or anti-guinea pig antibodies conjugated to peroxidase were added as appropriate. The plates were read using an ELISA reader at 405 nm. (B) Mixtures of serial dilutions of guinea pig serum were incubated with equal amounts of BRoVA. The mixture was incubated with a cell suspension to determine neutralization. The test was developed using a fluorescein isothiocyanate-labeled anti-RV polyclonal antiserum derived from a colostrum-deprived calf by hyperimmunization. Statistical analysis by ANOVA, and p < 0.001 (***).

Figure 9

Mice antibody titers induced by recombinant chimera. Total titers of IgG1 (A) and IgG2a (B). Groups are formed as in Figure 4 Group 1: 150 µL of PBS (Control), Group 2: 1 µg of EspB and 1 µg of Int280γ, Group 3: 2 µg of the chimera, Group 4: 10 µg of the chimera, Group 7: 1.10⁸ inactivated CFU of ETEC B41Arg and Salmonella Dublin, both expressing the chimera and Group 8: 1.10⁸ inactivated CFU of ETEC B41Arg and Salmonella Dublin, both expressing the chimera, plus 1.10⁷ FFU BRoVA UK and BCoVB Mebus.. Isotypes were determined in serum dilutions from various vaccinated groups using an indirect ELISA with a purified recombinant chimera as the antigen. Titers are expressed as geometric mean of each group (n = 5). Statistical analysis was performed by Bonferroni test, p < 0.01 (**) and p < 0.0001 (****).

Figure 10

Cytokine production by splenocytes from immunized mice. BALB/c mice were not immunized (control) or immunized with different vaccine formulations. Eighteen days after the last immunization, mice were sacrificed, and spleen cells were stimulated with purified recombinant chimera. After 72 h of culture, the concentrations of IFN-γ (A), IL-17A (B) and IL-5 (C) were determined in the culture supernatant by ELISA. The results are expressed as mean values (±standard error) of three experiments with 5 mice per group. Significant differences were analyzed for each cytokine between different vaccines for each stimulus, p < 0.05 (*) and p < 0.01 (**).