Obtention and characterization of the recombinant simian Interleukin-15 in Escherichia coli for 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 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.

KEYWORDS:

• CTLL-2 and Kit 225 cells
• E. coli
• in vitro proliferation assays
• protein
• recombinant siIL-15
• vaccine

Abbreviations:

• Ab: antibody
• Abs: antibodies
• Alum: Aluminum hydroxide
• ATCC: American Type Culture Collection
• bp: base pairs
• BSA: bovine serum albumin
• CENPALAB: National Center for Animal Breeding
• CIGB: Center for Genetic Engineering and Biotechnology
• CTLL: cytotoxic T-lymphocytes line
• E. coli: Escherichia coli
• EC₅₀: effective concentration at 50%
• EDTA: Ethylenediaminetetraacetic acid
• ELISA: enzyme-linked immunosorbent assay
• ESI: electrospray ionization
• FBS: fetal bovine serum
• HPLC: high-performance liquid chromatography
• huIL-15: human IL-15
• ID₅₀: half-inhibitory dilution
• IEX: ion exchange
• IFA: incomplete Freund’s adjuvant
• IL: Interleukin
• IL-15Rα: Interleukin-15 receptor α
• LB: Luria broth
• mhIL-15: modified human IL-15
• MS: mass spectrometry
• MTT: (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide
• NCBI: National Center for Biotechnology Information
• NHP: non-human primate
• OD: optical density
• PBMC: peripheral blood mononuclear cells
• PBS: phosphate-buffered saline
• RA: rheumatoid arthritis
• RP: reverse phase
• RT-PCR: reverse transcription-polymerase chain reaction
• SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
• siIL-15: simian IL-15
• TFA: trifluoroacetic acid

Acknowledgments

The authors express their deep gratitude to Dr. Yannick Jacques and Dr. Doreen Cantrell who provided the Kit225 human cell line. This acknowledgment is also extended to Yolanda Gomez, Marylens Hernadez, Maritza Arcia, and Carlos Manlio Garcia for their careful reading and reviewing of the manuscript; as well as to Mario Riera for the graphic assistance with Figure 1.

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Funding

This work was supported by the Center for Genetic Engineering and Biotechnology of Havana [Grant Number Not applicable].