Evaluation of Mycobacterium avium subsp. paratuberculosis isocitrate lyase (IcL) and ABC transporter (BacA) knockout mutants as vaccine candidates

Abstract

There has been little success in controlling Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, due to suboptimal diagnostics and the ineffectiveness of available vaccines. By knocking out BacA and IcL, genes required for MAP survival in dairy calves, two live-attenuated vaccine candidates were created. This study evaluated the host-specific attenuation of MAP IcL and BacA mutants in mouse and calf models, as well as the elicited immune responses. Deletion mutants were generated in MAP strain A1-157 through specialized transduction and found viable in vitro. First, the mutants' attenuation and elicited cytokine secretion were assessed in a mouse model, 3 weeks after intraperitoneal inoculation with MAP strains. Later, vaccine strains were assessed in a natural host infection model where calves received 10⁹CFU oral dose of MAP wild-type or mutant strains at 2 weeks old. Transcription levels of cytokines in PBMCs were evaluated at 12-, 14-, and 16-weeks post-inoculation (WPI) and MAP colonization in tissue was assessed at 4.5 months after inoculation. Whereas both vaccine candidates colonized mouse tissues similarly to wild-type strain, both failed to persist in calf tissues. In either mouse or calf models, gene deletion did not reduce immunogenicity. Instead, inoculation with ΔBacA induced a greater upregulation of proinflammatory cytokines than ΔIcL and wild-type in both models and a greater expansion of cytotoxic and memory T-cells than uninfected control in calves. ΔBacA and wild-type strains significantly increased secretion of IP-10, MIG, TNFα, and RANTES in mice serum compared to uninfected control. This agreed with upregulation of IL-12, IL-17, and TNFα in calves inoculated with ΔBacA at all time points. The ΔBacA also gave rise to greater populations of CD4+CD45RO+, and CD8+ cells than uninfected control calves at 16 WPI. Low survival rate of MAP in macrophages co-incubated with PBMCs isolated from the ΔBacA group indicated that these cell populations are capable of killing MAP. Overall, the immune response elicited by ΔBacA is stronger compared to ΔIcL and it is maintained over two different models and over time in calves. Further investigation is warranted to evaluate the BacA mutant's protection against MAP infection as a live attenuated vaccine candidate.

Keywords: Mycobacterium avium subsp. paratuberculosis; control programs; dairy calves; immune responses; live-attenuated vaccines; vaccine development.

Figure 1

Summary of the experimental methods used to create and evaluate MAP IcL and BacA mutants in mouse, calf, and ex-vivo trials. Illustrations were created in Biorender.

Figure 2

PCR primer schematic for MAP recombination screening.

Figure 3

MAP quantification in mice (n=14/group) liver and spleen, three weeks after intraperitoneal infection with 5×10⁸ CFU of MAP strains. F57 qPCR was employed to quantify genomic equivalents (GE) of MAP in 1 gram of tissue. The difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test.

Figure 4

Analysis of cytokine secretion in mice (n=14/group) serum three weeks after intraperitoneal infection with 5×10⁸ CFU of MAP wild-type and mutant strains. The uninfected control received PBS. The secretion level of cytokines in the serum were measured by Luminex assay. The difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test. * P≤0.05; ** P≤0.01; *** P≤0.001.

Figure 5

(A-C) Lymphocyte proliferation after stimulation of PBMCs with PPDa for 24 hours. PBMCs were isolated from all mutants and control groups at 12-, 14-, and 16- weeks post-inoculation and they were stimulated with MAP antigen for 24 hours. Flow cytometry was employed to evaluate populations of proliferated immune cells. The difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test. (D-F) Changes in cell populations within a group over time. The difference between timepoints were evaluated by one-way ANOVA followed by Tukey-Kramer test. Significant difference between groups (P value> 0.05) is indicated by lines above bars.

Figure 6

Cytokine expression within each group after stimulation of PBMCs with PPDa at 12-, 14-, and 16-weeks post inoculation. The difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test.

Figure 7

Cytokine expression analysis by RT-PCR, comparing groups at different timepoints. PBMCs were isolated from different groups (Uninfected =5, BacA=6, IcL=6, and wild-type=6). After 24 hours of in vitro stimulation with PPDa, RNA was extracted from PBMCs and reverse transcribed. RT-PCR was performed on cDNA, using 18s rRNA as a housekeeping gene and unstimulated samples as a calibrator. The data was analyzed by ΔΔct and the difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test. (B, D, F) Groups are color coded and numbers indicate animal IDs. Each group is clustered with a different ellipse. The vectors of the loading plots showing how strongly each variable influence a principal component, with their directions indicating how different variables are correlated with one another. The smaller the angel between two vectors, the more positively correlated they are. If they diverge and form a large angel close to 180 degrees, they are negative correlated.

Figure 8

(A) MAP viability assay comparing percentage of live MAP in infected macrophages alone, infected macrophages incubated with unprimed md-PBMCs, and infected macrophages incubated with primed md-PBMCs. (B) Viability assay comparing different treatments within groups. The difference among groups were assessed by one-way ANOVA and then further validated by Tukey-Kramer test. * P≤0.05; ** P≤0.01; *** P≤0.001.