Association of tuberculin-boosted antibody responses with pathology and cell-mediated immunity in cattle vaccinated with Mycobacterium bovis BCG and infected with M. bovis

Abstract

Vaccine development and our understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by definition of the immunological correlates of protection and/or pathology. In this study we analyzed humoral immune responses in Mycobacterium bovis BCG-vaccinated and control cattle (in particular, the relationship between the intradermal comparative tuberculin skin test and serum immunoglobulin G [IgG] responses) against a range of mycobacterial antigens (MPB59, MPB64, MPB70, MPB83, ESAT-6, CFP-10, Acr1, and PstS-1) by multiantigen print immunoassay and conventional enzyme-linked immunosorbent assay. Following M. bovis infection, the comparative tuberculin skin test strongly boosted IgG, IgG1, and IgG2 antibody responses, particularly against MPB83 and MPB70, in unvaccinated cattle but failed to boost these responses, or did so only weakly, in BCG-vaccinated calves. In addition, the skin test-induced increases in MPB83-specific IgG responses correlated positively with bacterial loads and ESAT-6-induced in vitro gamma interferon responses. In conclusion, both the negative correlation of skin test-enhanced MPB83-specific antibody responses with BCG-induced protection and their positive correlation with bacterial loads can serve as useful markers for vaccine efficacy after challenge.

FIG. 1.

Boost of MPB70 and MPB83 IgG1 responses due to tuberculin skin testing. Sera were collected before and 14 days after tuberculin skin testing, and IgG1 responses were determined by ELISA. OD₄₅₀ before skin testing were subtracted from those obtained post-skin test. Sera were diluted 1:100 for the ELISA. Filled circles, BCG-vaccinated, M. bovis-infected animals; open circles, unvaccinated, M. bovis-infected animals; horizontal bars, means. *, P < 0.001 (by an unpaired, two-tailed t test).

FIG. 2.

Positive correlations between skin test-induced increases in MPB83-specific IgG levels and disease severity (A), bacterial loads (B), and in vitro IFN-γ responses to ESAT-6 (C). Sera were collected before and 14 days after tuberculin skin testing, and IgG responses were determined by ELISA. OD₄₅₀ determined before skin testing were subtracted from those obtained post-skin test. (A) Increases in MPB83-specific IgG responses, determined by ELISA (serum dilution, 1:100), are shown in relation to total pathology scores (30). (B) Serum responses were correlated with total bacterial loads in the lymph nodes of vaccinated and unvaccinated animals (30). (C) Serum responses were correlated with in vitro IFN-γ production induced by ESAT-6. IFN-γ levels were determined by enzyme immunoassays of supernatants from ESAT-6-stimulated (5 mg/ml) whole-blood cultures performed 14 days post-skin test. Statistical analysis for all panels was conducted by using the Spearman rank test.