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A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.

Conditions were established to obtain depletion of T lymphocyte subsets in lymphoid tissues of calves by injection of mouse monoclonal antibodies to T cell antigens. Adverse reactions were avoided by injecting small quantities of antibody, until target cells had disappeared from blood. Two different mechanisms appeared to be responsible for elimination of the target cells. Rapid depletion of T cells was associated with complement-binding antibody isotypes (IgG2a, IgM), suggesting a complement-mediated mechanism. Clearance of T cells after several days was observed with a non complement-binding isotype (IgG1), suggesting phagocytosis or induction of apoptosis as possible mechanisms. Clearance of the cells in peripheral blood and spleen was obtained with 10-20 mg of anti-CD4 or anti-CD8, but almost ten times as much was needed to obtain depletion of the cells in lymph nodes and Peyer's patches. Depletion lasted for 12 days for CD4 T cells and 3 weeks for CD8 T cells. Successful and lasting depletion (at least 2 weeks) was also obtained with other T cell reagents, such as anti-CD2 and anti-WC1 (gamma/delta T cells). Although B lymphocytes could be removed by a complement-binding antibody, complete depletion of these cells only lasted for a few hours, probably because B cells regenerate faster than T cells. T cell function was severely inhibited when CD4+ T cells were depleted. Stimulation of T cells with foot and mouth disease viral antigen (FMDV) in vaccinated calves was non-existent after depletion. Even 2 months after restoration of normal CD4 T cell levels in blood, activity to FMDV was low. This suggested that the depleted T cells were replaced by naive cells.

Immunization of different mice strains with a recombinant fusion protein composed of the vector-encoded N-terminal leader peptide CroLac (containing lambda Cro and LacI fragments) and a part of the transmembrane protein of HIV-1 (gp41) led to a high anti-CroLac humoral immune response. A detailed analysis of this response revealed the presence of an immunodominant, linear B cell epitope localized near the C-terminus of the CroLac fragment. The immune response seemed to be biased towards this epitope since few or no monoclonal antibodies (mAb) could be generated against the remaining part of CroLac and the gp41 fragment. Upon removal of the immunodominant region from the fusion protein the immune response was redirected and spread over the previously non-immunogenic regions. Consequently, we report a model system in which an immunodominant B cell epitope biases the immune response away from less immunogenic epitopes on the same molecule.