Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A^202-347-SOD^1-72-Ag85B^173-330-74F^{1-148+669-786}), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum^® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum^® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum^® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Keywords: IFN-γ; IL-17; Johne’s disease; Mycobacterium avium subspecies paratuberculosis; gene expression; nanostring; recombinant MAP fusion protein particle vaccine; tuberculin skin test.

Figure 1

Antibody responses to recombinant antigen and PPDA in different vaccine groups. Mean serum antibody responses to (A), recombinant antigen (RA); and (B), PPDA in calves vaccinated with PBS, Silirum^®, rMAP fusion protein (Rec protein) and protein particles displaying MAP fusion protein (Protein particles) vaccines at weeks 0 and 3. Timing of vaccinations is indicated by arrows. Antibody titers were measured in sera before vaccination and after vaccination at weeks 3, 6, 9, and 12 using ELISA. Significance differences (P < 0.05) in antibody responses between different vaccine groups are indicated by different letters, while the same letter indicates no significant differences.

Figure 2

Antigen-specific cell-mediated immune responses in the vaccinated animals. Mean (+ SE) (A), IFN-γ and (B), IL-17A responses in the animals vaccinated with PBS; Silirum^®; recombinant MAP fusion protein (Rec protein); and protein particles displaying MAP fusion protein (Protein particles). Whole blood from the vaccinated calves at weeks 0, 3, 6, 9 and 12 were stimulated in vitro with PBS, PPDA, or RA. IFN-γ and IL-17 levels were measured by ELISA. Results for IFN-γ and IL-17A cytokine levels are presented as difference obtained by subtracting values of PBS- from PPDA- or RA-induced IFN-γ and IL-17A cytokines. Significance indicated as *, P < 0.05 compared with the respective treatment in the PBS vaccinated animals.

Figure 3

Expression of immune responsive genes in animal vaccinated with PBS (P), Silirum^® (S), recombinant MAP protein (RP), and MAP fusion protein particles (PP). Leukocytes were stimulated in vitro with either media alone, PPDA or RA at a final concentration of 10 μg/mL for 24 h. Results are presented as ratios of fold-change (antigen stimulation/media alone) for each gene at weeks 6, 9 and 12 over expression at week 0. The data was log2 transformed for statistical analysis and statistical significance was calculated by comparing ratios of antigen stimulation/media alone of all the vaccine groups and week 0 expression of the PBS control group. Statistical significance was calculated compared with the PBS group (* = P < 0.05; ** = P < 0.01; *** = P < 0.001). Note: No responses were measured in Silirum^®-vaccinated animals at week 12 due to RNA being lost during sample preparation.

Figure 4

Network plot showing Pearson correlations and canonical correlations between differentially expressed genes, antibody levels and T cell-mediated cytokine responses in cattle vaccinated with rMAP fusion protein at weeks 0, 6, 9 and 12. Genes are displayed as grey circles, gene categories are displayed as green rectangles and the three central circles colored pink, yellow and orange are, antibody, IL-17A (pg/mL), and IFN-γ (pg/mL) responses to RA, respectively. Thick black lines indicate the functional group that the genes belong to. Thick red and blue lines indicate canonical correlations above |0.6| and thin red lines indicate significant (P < 0.05) Pearson correlations between genes. Red lines indicate positive associations and correlations, while blue lines indicate negative associations and correlations. Darker line colors indicate stronger associations and correlations.

Figure 5

Comparative cervical tuberculin skin test in vaccinated animals. Skin thickness was measured before and at 72 h after injecting 0.1 mL of PPDA or PPDB intradermally at the side of the neck. Mean is represented by ‘─’ for each group. Significance is represented by between different groups. Statistical significance was calculated compared with the PBS control group (* = P < 0.05; ** = P < 0.01).