Obtention and characterization of the recombinant simian Interleukin-15 inEscherichia colifor the preclinical assessment of an IL-15-based therapeutic vaccine

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 EC 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.

Immunogenicity profile in African green monkeys of a vaccine candidate based on a mutated form of human Interleukin-15

“…IL-15 D8SQ108S, mhIL-15 and siIL-15 proteins were expressed in E. coli and purified following the procedure previously described for obtaining siIL-15 [ 27 ]. Briefly, the proteins expressed in the insoluble fraction were solubilized in an 8 M urea solution.…”

“…After a washing step, IL-15 containing fractions were eluted and the collected sample was applied to a C 4 column (1 × 25 cm, 10 µm, Vydac, USA) at a flow of 1 mL/min. Proteins were separated using a mobile phase containing solution A (0.1% TFA in water) and solution B (0.1% TFA in acetonitrile), using the same gradient as previously described [ 27 ]. Protein separations were monitored at 226 nm.…”

“…To evaluate whether the sera from immunized monkeys recognize the commercial huIL-15 and siIL-15, ELISA assays were performed as previously described by Rodríguez-Álvarez et al [ 19 , 27 ]. Briefly, wells were coated with 1 µg/mL of huIL-15 or siIL-15 in PBS for 16 h at 25 °C, and the pool of sera per group, corresponding to 15 days after the second and third immunizations, was evaluated in fixed dilution, 1:4000.…”

“…The effect of immune sera on the CTLL-2 cells proliferation induced by huIL-15, siIL-15 and huIL-2 was assessed using the CTLL-2 bioassay as previously described [ 19 , 27 , 44 ]. Briefly, 5 × 10 3 cells per well were cultured for 72 h with 300 pg/mL of huIL-15 or siIL-15 and serial dilutions 1:2 (starting dilutions 1:100 or 1:25) of the pool of sera per group collected 15 days after the second and third immunizations.…”

“…Therefore, this work was aimed to assess the immunogenicity in African green monkeys (AGM) of the vaccine candidate based on IL-15 D8SQ108S, as a potential strategy for treating autoimmune disorders involving IL-15 overexpression. Previously, IL-15 D8SQ108S was expressed in Escherichia coli and purified following the same procedure previously described for obtaining the recombinant simian IL-15 (siIL-15) [ 27 ]. In this article, the biological activity of the purified protein was determined in the CTLL-2 cell proliferation assay.…”