ABSTRACT
Recombinant simian IL-15 (siIL-15) was obtained for the preclinical assessment of an anti-human IL-15 vaccine. For this purpose, the cDNA from peripheral blood mononuclear cells of a Macaca fascicularis monkey was cloned into a pIL-2 vector. The siIL-15 was expressed in Escherichia coli strain W3110 as an insoluble protein which accounted for 13% of the total cellular proteins. Inclusion bodies were solubilized in an 8 M urea solution, which was purified by ion exchange and reverse phase chromatography up to 92% purity. The protein identity was validated by electrospray ionization-mass spectrometry, confirming the presence of the amino acids which distinguish the siIL-15 from human IL-15. The purified siIL-15 stimulates the proliferation of cytotoxic T-lymphocytes line (CTLL)-2 and Kit 225 cells with EC50 values of 3.1 and 32.5 ng/mL, respectively. Antisera from modified human IL-15-immunized macaques were reactive to human and simian IL-15 in enzyme-linked immunosorbent assays. Moreover, the anti-human IL-15 antibodies from immune sera inhibited siIL-15 activity in CTLL-2 and Kit 225 cells, supporting the activity and purity of recombinant siIL-15. These results indicate that the recombinant siIL-15 is biologically active in two IL-15-dependent cell lines, and it is also suitable for the preclinical evaluation of an IL-15-based therapeutic vaccine.
Subject(s)
Interleukin-15/genetics , Macaca fascicularis/genetics , Vaccines, Synthetic/genetics , Animals , Cell Line , Cloning, Molecular/methods , Escherichia coli/genetics , Humans , Interleukin-15/immunology , Macaca fascicularis/immunology , Mice , Recombinant Proteins/genetics , Recombinant Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, Synthetic/immunologyABSTRACT
BACKGROUND: Interleukin-15 is an immunostimulatory cytokine overexpressed in several autoimmune and inflammatory diseases such as Rheumatoid Arthritis, psoriasis and ulcerative colitis; thus, inhibition of IL-15-induced signaling could be clinically beneficial in these disorders. Our approach to neutralize IL-15 consisted in active immunization with structurally modified human IL-15 (mhIL-15) with the aim to induce neutralizing antibodies against native IL-15. In the present study, we characterized the antibody response in Macaca fascicularis, non-human primates that were immunized with a vaccine candidate containing mhIL-15 in Aluminum hydroxide (Alum), Montanide and Incomplete Freund's Adjuvant. RESULTS: Immunization with mhIL-15 elicited a specific antibodies response that neutralized native IL-15-dependent biologic activity in a CTLL-2 cell proliferation assay. The highest neutralizing response was obtained in macaques immunized with mhIL-15 adjuvanted in Alum. This response, which was shown to be transient, also inhibited the activity of simian IL-15 and did not affect the human IL-2-induced proliferation of CTLL-2 cells. Also, in a pool of synovial fluid cells from two Rheumatoid Arthritis patients, the immune sera slightly inhibited TNF-α secretion. Finally, it was observed that this vaccine candidate neither affect animal behavior, clinical status, blood biochemistry nor the percentage of IL-15-dependent cell populations, specifically CD56+ NK and CD8+ T cells. CONCLUSION: Our results indicate that vaccination with mhIL-15 induced neutralizing antibodies to native IL-15 in non-human primates. Based on this fact, we propose that this vaccine candidate could be potentially beneficial for treatment of diseases where IL-15 overexpression is associated with their pathogenesis.