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RP-HPLC chromatographic profile, identification, and purity analysis of purified siIL-15. (a) RP-HPLC chromatogram obtained after applying to a C 4 column the IL-15 containing peak from the IEX chromatography. The chromatogram represents the typical pattern of three independent experiments. (b) SDS-PAGE and (c) Western blot analysis of purified siIL-15 protein after the RP chromatography. Five hundred microliters of the peak collected at ∼96 min (indicated with an arrow) were concentrated; then 2 or 5 µg of the purified siIL-15 was loaded onto a 15% SDS-PAGE gel. Lanes 1 and 2: purified siIL-15 (2 and 5 µg, respectively). Lane 3: lysozyme was used as a molecular weight marker. An anti-huIL-15 monoclonal Ab was used to detect the protein of interest. Lane 1: purified siIL-15. Lane 2: recombinant huIL-15 from R&D was used as a positive control. Lane 3: lysozyme. (d) Determination of siIL-15 purity by RP-HPLC. 50 µg of purified siIL-15 was injected into a C 8 column, obtaining a principal peak with 92% of purity.
Source publication
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 E. coli strain W3110 as an insoluble protein which accounted for 13% of the tot...
Contexts in source publication
Context 1
... chromatogram of a semi-preparative RP-HPLC shows a peak at ∼96 min (Figure 4a). Samples collected at this time were assessed by SDS-PAGE and Western blot. ...
Context 2
... collected at this time were assessed by SDS-PAGE and Western blot. Figure 4b shows the unique band at 12.5 kDa, suggesting that recombi- 500 nant protein was obtained with almost 95% purity. The ident- ity of the protein was also verified by Western blot analysis, revealing that siIL-15 was the main protein in the preparation (Figure 4c). ...
Context 3
... 4b shows the unique band at 12.5 kDa, suggesting that recombi- 500 nant protein was obtained with almost 95% purity. The ident- ity of the protein was also verified by Western blot analysis, revealing that siIL-15 was the main protein in the preparation (Figure 4c). The purity of the protein obtained after the RP chromatography was additionally confirmed by analytical 505 RP-HPLC, using a C 8 column. ...
Context 4
... purity of the protein obtained after the RP chromatography was additionally confirmed by analytical 505 RP-HPLC, using a C 8 column. The retention time of siIL-15 was ∼13.1 min with a purity up to 92%, according to the analysis of the areas under the peaks (Figure 4d). This result revealed the presence of a single protein, without contaminat- ing protein in the sample. ...
Citations
... 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. ...
... 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. ...
Background
Interleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15.
Results
IL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys.
Conclusion
Immunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15.
... Additionally, the simian IL-15 (siIL-15) was obtained [12], in order to evaluate the effect of immune sera on the activity of siIL-15 in IL-15-dependent cell lines. It was demonstrated that, despite the high sequence homology in the amino acid sequence between hIL-15 and siIL-15, sera from monkeys immunized with mhIL-15 were able to neutralize the biological activity of siIL-15 in the CTLL-2 and Kit225 cells [12]. ...
... Additionally, the simian IL-15 (siIL-15) was obtained [12], in order to evaluate the effect of immune sera on the activity of siIL-15 in IL-15-dependent cell lines. It was demonstrated that, despite the high sequence homology in the amino acid sequence between hIL-15 and siIL-15, sera from monkeys immunized with mhIL-15 were able to neutralize the biological activity of siIL-15 in the CTLL-2 and Kit225 cells [12]. ...
Interleukin (IL)-15 is a pro-inflammatory cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory disease for which there is no effective therapy. In this report, we describe a novel vaccine based on active immunization with modified human IL-15 for the treatment of RA and others diseases related with IL-15 overexpression. The IL-15 obtained in E. coli exhibits a conformation of the disulfide bridges different to the one described for the native cytokine, which may favor the development of an immune response against this antigen. The results show that immunization with modified human IL-15 generates specific polyclonal antibodies against the cytokine in non-human primates, which suggests a rupture of B cells tolerance as consequence of immunization. These antibodies inhibited the biological activity of native IL-15 without affecting the human IL-2-induced proliferation of CTLL-2 cells, demonstrating the specificity of the antibodies by autologous IL-15. Additionally, we show that vaccination induces a regulated response of antibodies that neutralize the biological activity of simian IL-15, when aluminum hydroxide was used as adjuvant. The present work also provides the first safety elements of the anti-IL-15 vaccine in Macaca fascicularis monkeys, an animal model in which IL-15 shares a 97 % homology to the human molecule. This work received the Annual Award of the Cuban Academy of Sciences for the year 2017.
