Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni

Abstract

Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant) activity of the vaccine. The antigen (20-40 μg) was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

Fig 1. Quality properties of the NHC vaccine.

(A) The NHC protein was analyzed by SDS-PAGE in combination with PageBlue Protein Staining and by Western blotting using a C. jejuni 81116 FlaA flagellin-specific antibody. (B-E) NF-κB luciferase activity of HeLa57A cells expressing (B) chTLR5, (C) chTLR2t2/chTLR16, (D) chTLR21 and (E) chTLR4, after stimulation (5 h) with NHC flagellin or the respective positive controls. Results are expressed as fold increase in stimulated cells versus non-stimulated cells and represent the mean ± SEM of three independent experiments.

Fig 2. Determination of challenge dose of C. jejuni strain 81116.

Chickens were orally inoculated with the indicated dose of C. jejuni strain 81116. Four days post-inoculation the number of C. jejuni in the chicken ceca was determined by plate counting and expressed as colony forming units per gram cecal content (CFU/ g). Symbols represent individual animal bacterial loads (log₁₀ CFU/ g), and bars represent the averages.

Fig 3. Antigen-specific serum antibody responses.

NHC flagellin-specific antibody levels in the groups immunized with (A) 40 μg of NHC protein and (B) 20 μg of NHC protein, both compared to levels in the solvent injected control group. Antigen-specific IgY, IgM or IgA antibody levels were determined in 20-fold diluted sera collected from 12 day-old individual animals. Results are displayed as a box-and-whisker plot (S/R, sample to reference ratio). An asterisk indicates statistically significant differences (α < 0.05) between compared groups.

Fig 4. Kinetics of NHC-specific antibody levels following in ovo vaccination.

ELISA results demonstrating the NHC flagellin-specific reactivity of serially diluted sera collected from (A, C, E) 12 day-old and (B, D, F) 18 day-old chickens injected with 40 μg of NHC flagellin or solvent (control). Serum IgY (A, B), IgM (C, D) and IgA (E, F) antibody responses are indicated. Graphs represent the analysis of individual animals, displayed as a box-and-whisker plot. The amount of antigen-specific antibodies was expressed in a relation to the reference sera (S/R, sample to reference ratio). An asterisk indicates statistically significant differences (α < 0.05) between groups.

Fig 5. Western blot analysis of chicken sera.

Purified flagellins (FlaA, NHC) and whole cell lysates of C. jejuni 81116 (wild type and ΔflaAB) were analyzed by Western blotting. Pooled sera collected from 18 day-old chickens injected with (A) NHC flagellin (40 μg) or (B) solvent were used as probes and detected with anti-chicken IgY antibody. Molecular mass is indicated in kilodaltons (kDa). The arrowhead indicates the reactivity of the sera with the native C. jejuni FlaA flagellin that is absent in the ΔflaAB mutant strain.

Fig 6. Cecal colonization of C. jejuni and sIgA responses in vaccinated and control chickens.

(A) Chickens were challenged with C. jejuni at 18 day of age. One week post-inoculation (25 days of age) the number C. jejuni in the ceca was determined by plate counting (CFU/ g). Each symbol represents one chicken of the indicated groups, bars represent the mean number of log₁₀ CFU/ g per group. (B) Antigen-specific sIgA in 20-times diluted cecal content from 25 day-old chickens (one week after C. jejuni challenge) as determined by ELISA. Graphs represent the analysis of individual animals of each of the indicated groups, displayed as a box-and-whisker plot.