Characterization of early immune responses elicited by live and inactivated vaccines against Johne's disease in goats

Abstract

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic debilitating condition affecting ruminants causing significant economic losses to the dairy industry. Available inactivated vaccines are not effective in controlling the disease and vaccinated animals can continue to infect newly born calves. Recently, we have shown that a live-attenuated vaccine candidate (pgsN) is protective in goats and calves following challenge with virulent strains of M. paratuberculosis. To decipher the dynamics of the immune responses elicited by both live-attenuated and inactivated vaccines, we analyzed key immunological parameters of goats immunized through different routes when a marker-less pgsN vaccine was used. Within a few weeks, the inactivated vaccine triggered the formation of granulomas both at the site of inoculation and in regional lymph nodes, that increased in size over time and persisted until the end of the experiment. In contrast, granulomas induced by the pgsN vaccine were small and subsided during the study. Interestingly, in this vaccine group, histology demonstrated an initial abundance of intra-histiocytic mycobacterial bacilli at the site of inoculation, with recruitment of very minimal T lymphocytes to poorly organized granulomas. Over time, granulomas became more organized, with recruitment of greater numbers of T and B lymphocytes, which coincided with a lack of mycobacteria. For the inactivated vaccine group, mycobacterial bacilli were identified extracellularly within the center of caseating granulomas, with relatively equal proportions of B- and T-lymphocytes maintained across both early and late times. Despite the differences in granuloma-specific lymphocyte recruitment, markers for cell-mediated immunity (e.g., IFN-γ release) were robust in both injected pgsN and inactivated vaccine groups. In contrast, the intranasal live-attenuated vaccine did not elicit any reaction at site of inoculation, nor cell-mediated immune responses. Finally, 80% of animals in the inactivated vaccine group significantly reacted to purified protein derivatives from M. bovis, while reactivity was detected in only 20% of animals receiving pgsN vaccine, suggesting a higher level of cross reactivity for bovine tuberculosis when inactivated vaccine is used. Overall, these results depict the cellular recruitment strategies driving immune responses elicited by both live-attenuated and inactivated vaccines that target Johne's disease.

Keywords: Johne's disease; immunity; inactivated vaccine; live vaccine; paratuberculosis (MAP); vaccine.

Figure 1

Sketch for experimental design used in this study. Goat kids at 28 days of age were vaccinated by subcutaneous injection or intranasal and monitored for 6 months post vaccination. Two sacrifices were conducted at 3 weeks then at 6 months post vaccination. Feces, blood and saliva were collected once a month throughout the experiment timeline.

Figure 2

Genome-wide analysis of live attenuated vaccine pgsN. (A) Annotated genes on the aligned M. paratuberculosis ΔlipN showing from outer to inner; scaffold of M. paratuberculosis k10, coding DNA sequences (CDS) on forward, CDS on reverse, non-CDS features, non-synonym mutations on forward CDS, non-synonym mutations on reverse CDS, GC content and GC skew. (B) Magnification of short reads aligned to areas flanking the lipN gene to confirm successful knockout of lipN by the absence of aligned reads.

Figure 3

Safety of pgsN vaccine. (A) The average body temperatures at 1 day before vaccination, the first 3 days post vaccination as well as every month post vaccination. (B) The average weights for each group throughout the study timeline. (C) The average size of the granuloma at the site of injection. Bars represent the standard error asterisks indicate statistical significance between pgsN-SC and inactivated vaccines using Student t-test. **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001.

Figure 4

Histopathology of granuloma formation in goats after immunization with live-attenuated or inactivated vaccines at 3 weeks post vaccination. (A, B) Hematoxylin and eosin (H&E) stained sections from subcutaneous granulomas at the site of injection of SC-vaccinated goats demonstrating granulomatous inflammation (A) to granuloma formation (B) (bars = 500 μm). (A) Top (H&E) inset shows sheets of epithelioid macrophages, while the bottom (acid-fast stain) inset shows myriad intra-histiocytic acid-fast bacilli (AFB). (B) Top (H&E) inset displays degenerate neutrophils (asterisk) at the center of the granuloma, with intralesional bacilli in both H&E and acid-fast stain insets. (C, D) Immunohistochemical (IHC) staining of the sections in (A, B) with arrows pointing to minimal (C) or small (D) numbers of CD3 + T lymphocytes (bars are 20 & 50 μm, respectively). (E, F) IHC staining of the sections in (A, B) with arrows pointing to small to moderate numbers of CD20+ B lymphocytes in F, with no detectable B lymphocytes in E (bars = 20 μm).

Figure 5

Histopathology of granuloma formation in goats after immunization with live attenuated or inactivated vaccine at 6 months post vaccination. (A, B) Hematoxylin and eosin (H&E) stained sections from subcutaneous granulomas at the site of injection of a single SC-vaccinated goat (no other goats in the live-attenuated cohort had grossly or histologically evident granulomas, while all goats in the inactivated vaccine group had subcutaneous granulomas). Arrows point to lymphoid nodular aggregates at the periphery of granulomas, with peri-granuloma lymphocyte recruitment further highlighted by the top (H&E) inset (A) (bar = 100 μm) and top (H&E) inset (B) (bar =50 μm). The bottom (acid-fast stain) inset in (A) shows lack of AFB in subcutaneous granulomatous areas (A) in the single pgsN-vaccinated animal; in contrast, extracellular bacilli are present within the caseating center of subcutaneous granulomas in the inactivated vaccine group [(B); acid-fast stain, bottom inset]. (C, D) Immunohistochemistry (IHC) staining of the sections in (A, B) with arrows highlighting CD3 + T lymphocytes located at the periphery of granulomas (bars = 50 μm). (E, F) IHC staining of the sections in (A, B) with arrows pointing to CD20 + B lymphocytes forming large nodular aggregates that peripheralize granulomas (bars = 500 and 50 μm, respectively).

Figure 6

Immune responses to vaccination. Evaluation of cell Mediated Immunity as measured by reaction to intradermal skin test at (A) 3 and (B) 6 months post vaccination (5 animals/group). Each point in (A, B) represents one animal's difference in skin thickness at the PPD site of injection at 0 and 72 h post injection. Purified protein derivative from both M. paratuberculosis (PPD-j) and M. bovis (PPD-b) were used to inject animals. Bars represent the standard error measurements for each group. (C) Measurement of IFN-γ levels in vaccinated goat groups. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stimulated with PPD-j for 72 h. IFN-γ levels in culture supernatants were determined using a Bovigam ELISA kit. Data are expressed as an ELISA index, with error bars represent the standard error. (D) Humoral Immune responses as measured by commercial ELISA from sera collected from immunized animals. Asterisks indicate statistical significance between each group immunized with subcutaneous injection compared to PBS using ANOVA. *, p-value < 0.05; **, p-value < 0.01; ****, p-value < 0.0001. In (D) only inactivated vaccine gave significant difference.