Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys.

Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies.

Immunotoxic effects of cyclophosphamide and cyclosporine in the dog.

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation.

Development of a Delayed-Type Hypersensitivity (DTH) Model in the Cynomolgus Monkey.

Although a T-dependent antibody response (TDAR) assay is generally recommended as the first-line immune function assay in nonclinical immunotoxicity evaluation, second-line assays such as delayed-type hypersensitivity (DTH) to measure cell-mediated responses can provide helpful additional information. In this study, male Cynomolgus monkeys were injected intramuscularly either once or twice with 1 mg Keyhole Limpet Hemocyanin (KLH) or twice with a commercially available tetanus vaccine (40 IU tetanus toxoid + 0.06 mg aluminum hydroxide). All animals were subsequently challenged by intradermal injections of the same antigen or aluminum hydroxide after 4, 6 and 8 weeks. Clinical reactions at the injection sites were scored 24, 48 and 72 h post challenge. Skin biopsies were taken on completion of the observation period after each challenge for standard histological examination and immunolabeling using CD3 (T lymphocytes), CD19 (B lymphocytes) and CD68 (macrophages) antibodies. Tetanus toxoid induced stronger clinical reactions than KLH, whereas aluminum hydroxide induced no clinical reaction. Perivascular mononuclear cell infiltrates, a histopathological finding consistent with a DTH reaction, were seen after all challenges with tetanus toxoid or KLH, but not with aluminum hydroxide. Immunohistochemistry evidenced the presence of T lymphocytes and macrophages within these infiltrates. These results suggest that tetanus toxoid adjuvanted with aluminum hydroxide can induce a consistent DTH response for use as a model of cell-mediated response in Cynomolgus monkeys.