A new class of highly antigenic, MHC-II–restricted mycobacterial lipopeptides that are recognized by CD4-positive T lymphocytes of Mycobacterium tuberculosis–infected humans has recently been described. To investigate the relevance of this novel class of mycobacterial Ags in the context of experimental bacille Calmette-Guérin (BCG) vaccination, Ag-specific T cell responses to mycobacterial lipid and lipopeptide-enriched Ag preparations were analyzed in immunized guinea pigs. Lipid and lipopeptide preparations as well as complex Ag mixtures, such as tuberculin, mycobacterial lysates, and culture supernatants, all induced a similar level of T cell proliferation. The hypothesis that lipopeptide-specific T cells dominate the early BCG-induced T cell response was corroborated in restimulation assays by the observation that Ag-expanded T cells specifically responded to the lipopeptide preparation. A comparative analysis of the responses to Ag preparations from different mycobacterial species revealed that the antigenic lipopeptides are specific for strains of the M. tuberculosis complex. Their intriguing conservation in pathogenic tuberculous bacteria and the fact that these highly immunogenic Ags seem to be actively released during in vitro culture and intracellular infection prompt the urgent question about their role in the fine-tuned interplay between the pathogen and its mammalian host, in particular with regard to BCG vaccination strategies.

The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. Aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future. To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.

Bovine neonatal pancytopenia (BNP) was a vaccine-induced alloimmune disease observed in young calves and characterized by hemorrhages, pancytopenia, and severe destruction of the hematopoietic tissues. BNP was induced by alloreactive maternal antibodies present in the colostrum of certain cows vaccinated with a highly adjuvanted vaccine against bovine viral diarrhea. Bioprocess impurities, originating from the production cell line of the vaccine, are likely to have induced these alloreactive antibodies. One prominent alloantigen recognized by vaccine-induced alloantibodies is highly polymorphic bovine major histocompatibility complex class I antigen (bovine leukocyte antigen 1—BoLA I). Aim of this study was to define the fine specificity of BNP-associated anti-BoLA I alloantibodies. In total, eight different BoLA I alleles from the production cell line were identified. All genes were cloned and recombinantly expressed in murine cell lines. Using these cells in a flow cytometric assay, the presence of BoLA I specific alloantibodies in BNP dam sera was proven. Three BoLA I variants were identified that accounted for the majority of vaccine-induced BoLA I reactivity. By comparing the sequence of immunogenic to non-immunogenic BoLA I variants probable minimal epitopes on BoLA I were identified. In general, dams of BNP calves displayed high levels of BoLA I reactive alloantibodies, while vaccinated cows delivering healthy calves had significantly lower alloantibody titers. We identified a subgroup of vaccinated cows with healthy calves displaying very high alloantibody titers. Between these cows and BNP dams no principle difference in the BoLA I reactivity pattern was observed. However, with a limited set of dam-calf pairs it could be demonstrated that serum from these cows did not bind to BoLA I expressing leukocytes of their offspring. By contrast, when testing cells from surviving BNP calves with the corresponding dam’s serum there was significant binding. We therefore conclude that predominantly highly alloreactive cows are at risk to induce BNP and it depends on the paternally inherited BoLA I whether or not the calf develops BNP.

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The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. The aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future. To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.