The project’s main idea is to study the effect of recombinant interleukin 15 (IL-15) on increasing immunity against bovine leukemia, both alone and with anti-CTLA-4 and anti-PD-L1 antibodies. IL-15 increases the proliferation of NK and CD8 + T cells and enhances the immune system’s effectiveness against bovine leukemia. CTLA-4 and PD-L1 receptors are immune system checkpoints, the increase of which causes the progression of chronic viral infections. In this regard, there is a hypothesis that treatment with recombinant IL-15, anti-PD-L1, and anti-CTLA-4 antibodies will increase the activity of NK and CD8 + T cells and the production of cytokines in animals with leukemia.
In the current situation with bovine leukemia virus, scientific and technological needs arise to develop new practical approaches to treating the infection. One promising molecule proposed for the treatment of cancer is IL-15. This molecule increases the proliferation of NK and CD8 + T cells and may help enhance the immune system’s effectiveness against bovine leukemia. Together with antibodies against CTLA-4 and PD-L1 receptors, IL-15 can effectively block the progression of chronic viral infections. In this regard, studying the complex use of IL-15, anti-PD-L1, and anti-CTLA-4 antibodies to increase NK and CD8 + T cells and cytokine concentrations is an urgent problem.
The project’s novelty lies in studying the possibility of using IL-15 in combination with anti-CTLA-4 and anti-PD-L1 antibodies to increase cellular immunity in cows infected with bovine leukemia virus. IL-15 is of great importance for enhancing the proliferation and effector functions of NK cells. At the same time, IL-15 does not increase the concentration of T-reg cells. In clinical trials, intravenous administration of IL-15 resulted in increases in NK cells, CD56 NK cells, and CD8 T cells. However, monotherapy with IL-15 drugs was ineffective due to the action of the PD-L1 and CTLA-4 immunological checkpoints and the lack of tumor-specific targeting of NK cells. Combining anti-PD-L1 antibodies with IL-15 had a dual effect in mouse tumor models and further enhanced the antitumor activity of the PD-1 agonist. The use of IL-15 in combination with antibodies to PD-L1 and CTLA-4 in carcinoma models significantly increased the survival of mice.
The goal of the project is to study the combined effect of IL-15 and antibodies against CTLA-4 and PD-L1 receptors on increasing the activity of immunity against bovine leukemia
As a result of the study, an E. coli strain producing recombinant IL-15 will be obtained. Protocols for protein isolation, purification and renaturation will be developed. IL-15 plays a crucial role in developing and proliferating NK and memory CD8+ T cells. The effect of complex use of IL-15, anti-CTLA-4 and anti-PD-L1 antibodies on the level of expression of Bcl2, STAT3, STAT5, Cpt1a genes and IFN-γ in peripheral blood mononuclear cells of cattle will be studied. The effect of continuous exposure of IL-15, anti-CTLA-4 and anti-PD-L1 antibodies on the level of immunological response against bovine leukemia virus in infected cows will be studied.
Mukantayev Kanatbek Naizabekovich, Doctor of Biological Sciences, Associate Professor, h-index 4 (Author ID Scopus: 57211138932).
Tursunov K.A., PhD., Senior researcher, h-index 3, Scopus Author ID: 57193579180; Researcher ID: N-6319-2017; ORCID: 0000-0001-8260-2563.
Adish Zh., PhD doctoral student in biology, research fellow, h-index 2 Scopus Author ID: 57202535857; Researcher ID: AAW-7200-2020; ORCID: 0000-0001-9527-8774.
Kanayev D.B., Master of Biology, Researcher, ResearcherID: N-6950-2017; ORCID: 0000-0001-9569-9034; Author ID Scopus: 27864.
Nurtleu M., PhD doctoral student in biology, Junior Researcher, h-index 1, Scopus Author ID: 57202536508; Researcher ID: N-6297-2017; ORCID: 0000-000-1299-8782.
1) Malika, N., Zhansaya, A., Kasym, M., Kanat, T., Yerlan, R., Kanatbek, M. Analysis of Antibody Induction by Macrophages Treated Ex Vivo with Human Proteins in Mice (2023) Reports of Biochemistry and Molecular Biology, 11(4), P.694-701. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85153291601&partnerID=40&md5=015f2c907ce425a4b8c094005c244bf
2) Tursunov, K., Tokhtarova, L., Kanayev, D., Mustafina, R., Mukantayev, K. Effect of thioredoxin on the immunogenicity of the recombinant P32 protein of lumpy skin disease virus (2022) Veterinary World, 15(10), P.2384-2390. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85140918029&doi=10.14202%2fvetworld.2022.2384-2390&partnerID=40DOI: 10.14202/vetworld.2022.2384-2390
3) Mukantayev, K., Kanayev, D., Zhumabekova, S., Shevtsov, A., Tursunov, K., Mukanov, K., Ramankulov, Y. Optimization of polymerase chain reaction for the identification of Roe deer, Saiga, and Siberian stag living in Kazakhstan (2022) Veterinary World, 15(8), P.2067-2071. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85138941681&doi=10.14202%2fvetworld.2022.2067-2071&partnerID=40DOI: 10.14202/vetworld.2022.2067-2071
4) Bulashev, A.K., Ingirbay, B.K., Mukantayev, K.N., Syzdykova, A.S. Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle (2021) Veterinary World, 14(8), P.2187-2196. Citations – 4. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85116104005. DOI: 10.14202/vetworld.2021.2187-2196
5) Sotnikov, D.V., Barshevskaya, L.V., Zherdev, A.V., Eskendirova, S.Z., Mukanov, K.K., Mukantayev, K.K., Ramankulov, Y.M., Dzantiev, B.B. Immunochromatographic system for serodiagnostics of cattle brucellosis using gold nanoparticles and signal amplification with quantum dots (2020) Applied Sciences (Switzerland), 10(3). Citations – 8. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85081584093. doi=10.3390%2fapp10030738
6) Mukantayev, K., Kairova, Z., Tursunov, K., Shustov, A., Zhumabekova, S., Ramankulov, E., Mukanov, K. Recombinant expression and purification of a pathogen-specific murein hydrolase lysin from γ-bacteriophage of Bacillus anthracis (2019) Current Topics in Peptide and Protein Research, 20, P.41-49. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85080894201&partnerID=40&md5=1337be9c346e87a8efb3f74e5062273
7) Sotnikov, D.V., Berlina, A.N., Zherdev, A.V., Eskendirova, S.Z., Mukanov, K.K., Ramankulov, Y.M., Mukantayev, K.N., Dzantiev, B.B. Comparison of Three Schemes of Quantum Dots-Based Immunochromatography for Serodiagnosis of Brucellosis in Cattle (2019) ARPN Journal of Engineering and Applied Sciences, 14(11), P.3711-3718. Citations – 6. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85077886794. doi=10.36478%2fJEASCI.2019.3711.3718
8) Mukanov, K.K., Adish, Z.B., Mukantayev, K.N., Tursunov, K.A., Kairova, Z.K., Kaukabayeva, G.K., Kulyyassov, A.T., Tarlykov, P.V. Recombinant expression and purification of adenocarcinoma gpr161 receptor (2019) Asia-Pacific Journal of Molecular Biology and Biotechnology, 27(4), P.85-95. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85074484552&doi=10.35118%2fapjmbb.2019.027.4.10&partnerID=40&mDOI: 10.35118/apjmbb.2019.027.4.10
9) Barshevskaya, L.V., Sotnikov, D.V., Zherdev, A.V., Khassenov, B.B., Baltin, K.K., Eskendirova, S.Z., Mukanov, K.K., Mukantayev, K.K., Dzantiev, B.B. Triple immunochromatographic system for simultaneous serodiagnosis of bovine brucellosis, tuberculosis, and leukemia (2019) Biosensors, 9(4). Citations – 2. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85072779454&doi=10.3390%2fbios9040115&partnerID=40&md5=a8180DOI: 10.3390/bios9040115
10) Sotnikov, D.V., Berlina, A.N., Zherdev, A.V., Eskendirova, S.Z., Mukanov, K.K., Ramankulov, Y.M., Mukantayev, K.N., Dzantiev, B.B. Immunochromatographic serodiagnosis of brucellosis in cattle using gold nanoparticles and quantum dots (2019). International Journal of Veterinary Science, 8(1), P.28-34. Citations – 7. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85063189960&partnerID=40&md5=c9ba666b2eb9772380eff393ec05e6fd
11) Bulashev, A., Jakubowski, T., Mukantayev, K., Tursunov, K., Kiyan, V., Zhumalin, A. Using combined recombinant protein in the diagnosis of bovine brucellosis (2018) Medycyna Weterynaryjna, 74(3), P.193-198. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85045322253&doi=10.21521%2fmw.6079&partnerID=40&md5=3a572d6DOI: 10.21521/mw.6079
12) Mukantayev, K., Tursunov, K., Ingirbay, B., Adish, Z., Azhibayeva, M., Kairova, Z., Ramankulov, E., Mukanov, K., Shustov, A. Immunochromatographic assay for the foot-and-mouth disease utilizing recombinant nonstructural proteins 2C, 3A, 3B and 3D (2018) Bulgarian Journal of Agricultural Science, 24(3), P.489-496. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85048693752&partnerID=40&md5=ac449c9e39df7550359671fad4b61d5
13) Mukantayev, K., Tursunov, K., Raimbek, G., Shustov, A., Begaliyeva, A., Ingirbay, B., Mukanov, K., Ramanculov, E. Immunochromatographic assay for diagnosis of bovine leukaemia virus infection in cows using the recombinant protein gp51 (2018) Veterinarija ir Zootechnika, 76(98), P.34-40. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85047502402&partnerID=40&md5=a120bf3a11110d66b0c3e18c9402db8
14) Tursunov, K., Begaliyeva, A., Ingirbay, B., Mukanov, K., Ramanculov, E., Shustov, A., Mukantayev, K. Cloning and expression of fragment of the rabies virus nucleoprotein gene in Escherichia coli and evaluation of antigenicity of the expression product (2017) Iranian Journal of Veterinary Research, 18(1), P.36-42. Citations – 4. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85015014330&partnerID=40&md5=4757d6d8057b200aec87f468444442
15) Zhansaya, A., Malika, N., Boris, D., Kanat, T., Kanatbek, M., Yerlan, R., Kasym, M. Expression of Recombinant CTLA-4 and PD-L1 Proteins Fused with Thioredoxin, and Determination of Their Ligand-Binding Activities (2022) Reports of Biochemistry and Molecular Biology, 11(2), P.310-319. Citations – 2. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85135595075&partnerID=40&md5=5bec56ec7fd5691e7fcdda19b927a45
16) Zhansaya, A., Kanatbek, M., Kanat, T., Bakhytkali, I., Darkhan, K., Arman, K., Pavel, T., Kasym, M., Yerlan, R. Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor (2020) Reports of Biochemistry and Molecular Biology, 8(4), P.347-357. Citations – 2. https://www.scopus.com/inward/record.uri?eid=2-s2.0-85104813247&partnerID=40&md5=468810d4b6680952333180ff845bdfb
17) Escherichia coli microorganism strain B834/pET15/3A is a producer of the recombinant non-structural protein 3A of the foot-and-mouth disease virus. Innovative patent No. 25095. Mukantaev K.N., Tursunov K., Lazarev V.N., Levitsky S.A., Kharlampieva D.D., Kushcheva N.A., Baltin K.K., Mukanov K.K., Ramankulov E.M.
18) A hybrid strain of cultured animal cells Mus Musculus L., a producer of monoclonal antibodies to the recombinant antigen p24 of the bovine leukemia virus. Copyright certificate No. 73446. Mukantaev K.N., Bakirova G.A., Belyalova A.R., Zhylkibaev A.A., Baltin K.K., Mukanov K.K., Ramankulov E.M.
19) Escherichia coli microorganism strain B834/pET32/TpN17 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN17. Copyright certificate No. 73335. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
20) The microorganism strain Escherichia coli B834/pET32/TpN47 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN47. Copyright certificate No. 73328. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
21) Escherichia coli microorganism strain B834/pET32/TpN15 is a producer of the recombinant membrane lipoprotein Treponema pallidum TpN15. Author’s certificate No. 73314 1 Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
22) The microorganism strain Escherichia coli B834/pET32/TprK is a producer of the recombinant membrane lipoprotein Treponema pallidum TprK. Copyright certificate No. 73307. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
23) The microorganism strain Escherichia coli B834/pET32/Tp0453 is a producer of the recombinant membrane lipoprotein Treponema pallidum Tp0453. Copyright certificate No. 73321. Mukantaev K.N., Bakirova G.A., Lazarev V.N., Levitsky S.A., Shkarupeta M.M., Mukanov K.K., Ramankulov E.M.
24) The strain of the microorganism Escherichia coli BL21/pET32/VP1 Asia is a producer of the recombinant VP1 antigen of the foot-and-mouth disease virus of the Asia type. Copyright certificate No. 88014. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Baidosova Sh.
25) Escherichia coli microorganism strain BL21/pET32/VP1 O producer of recombinant VP1 antigen of foot-and-mouth disease virus type O. Author’s certificate No. 88019. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Baidosova Sh.
26) Escherichia coli microorganism strain BL21/pET32/VP1 A producer of recombinant VP1 antigen of foot-and-mouth disease virus type A. Author’s certificate No. 90924. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Tursunov K., Begalieva A.
27) Escherichia coli microorganism strain BL21/E3.pET22/gp51 is a producer of the recombinant gp51 antigen of the bovine leukemia virus. Patent for invention No. 30998. Mukanov K.K., Ramankulov E.M., Shustov A.V., Mukantaev K.N., Tursunov K., Begalieva A.
28) The microorganism strain Escherichia coli BL21(DE3)/pET32/MPRV is a producer of the recombinant matrix protein of the rabies virus. Author’s ID No. 106743. Mukantaev K.N., Shustov A.V., Tursunov K.A., Inirbay B., Adish Zh., Ramankulov E.M., Mukanov K.K.
29) Escherichia coli microorganism strain BL21(DE3)/pET32/NPRV is a producer of recombinant rabies virus nucleoprotein. Author’s ID No. 101822. Mukantaev K.N., Shustov A.V., Tursunov K.A., Inirbay B., Adish Zh., Ramankulov E.M., Mukanov K.K.
2023
The amino acid sequence of the protein and the nucleotide sequence of the bovine IL-15 were analyzed. The amino acid sequence obtained from the PubMed GenBank database was used for analysis: AAA85130.1. Based on an analysis of literature data, the active site of a cytokine with a length of 166 amino acid residues was selected. Based on the selected amino acid sequence, a codon-optimized bovine IL-15 genes was developed for efficient expression in E. coli. Based on the developed nucleotide sequence, 16 oligonucleotides 60 pairs long were obtained. A fragment of the bovine IL-15 gene, 486 base pairs long, was synthesized under de novo conditions. As a result of the design, schemes of genetic constructs were developed based on pET28 and pET32 vectors, carrying the gene of a selected fragment of IL-15 with the correct reading frame. In the pET28 vector, the bovine IL-15 gene is inserted into the EcoRI and XhoI restriction sites. The expression region includes the 6His-Tag, thrombin site, interleukin, and 6His-Tag genes. The predicted molecular mass of the protein is 24 kDa. In the pET32 vector, the IL-15 gene is inserted at the NcoI and XhoI restriction sites. The expression region includes the thioredoxin protein, His-Tag, S-Tag, thrombin, and enterokinase genes, interleukin, and 6His-Tag. The predicted molecular mass of the protein is 35 kDa.
As a result of cloning the bovine IL-15 gene into pET28 and pET32 plasmids, we obtained the expression plasmids pET28/IL15bovine and pET32/IL15bovine. The resulting genetic constructs were transformed into E. coli BL21 cells by electroporation. Transformed cells were cultured on solid LB+kan or LB+amp medium at 37°C. As a result, colonies with cultural properties corresponding to E. coli were obtained. Screening of colonies by PCR showed that they contained a product 486 base pairs long, corresponding to the size of IL-15. Selected colonies were cultured in liquid LB+kan or LB+amp medium at 37°C. After centrifugation, the cells were resuspended in cryopreservation medium and frozen. Analysis of the resulting cell suspension of E. coli BL21/pET28/IL15bovine by SDS-PAGE showed the presence of a recombinant protein with a molecular weight of 24 kDa. Analysis of the resulting cell suspension of E. coli BL21/pET32/IL15bovine by SDS-PAGE showed the presence of a recombinant protein with a molecular weight of 35 kDa. To determine expression activity, recombinant strains E. coli BL21/pET28/IL15bovine and E. coli BL21/pET32/IL15bovine were incubated in LB medium at different concentrations of IPTG, different temperatures and incubation durations. It has been established that the optimal parameters for the expression of recombinant proteins are 0.2 mM IPTG, 37°C and 16-hour induction. These parameters were used to further isolate and purify recombinant bovine IL-15 (rIL15b). Purification of rIL15b was performed using Ni2+-NTA chromatography under denaturation conditions with 8 M urea. For correct refolding of rIL15b, a linear gradient of urea from 8 to 0 M was used in His-tag columns. Recombinant proteins were eluted with linear gradients of imidazole from 20 to 500 mmol/L.
Recombinant proteins were characterized by 12% SDS-PAGE, western blotting, and LC-MS/MS spectrometry. Western blotting of recombinant proteins produced by BL21/pET28/IL15bovine and BL21/pET32/IL15bovine cells with monoclonal antibodies against His-tag revealed protein bands with a molecular mass of 24 and 35 kDa, respectively. The LC-MS/MS spectral data of rIL15b ions were analyzed using the Mascot database. As a result of the analysis, the spectral ions corresponded to bovine IL-15 (Scor 157.9).
2024
The biological activity of rbIL-15 was determined by stimulating peripheral blood mononuclear cells (PBMCs) from cattle and detecting mRNA of factors such as Bcl2, STAT3, and STAT5. PBMCs were stimulated with various concentrations of purified recombinant IL-15 (1 µg, 500 ng, 250 ng, and 125 ng). Since STAT3 and STAT5 proteins are involved in cytokine signaling via the JAK-STAT pathway, the effect of IL-15 on the expression of STAT3 and STAT5 was studied using PCR by quantifying the DNA encoding these proteins. Complementary DNA (cDNA) synthesized from mRNA was analyzed using PCR, followed by agarose gel electrophoresis. Application of rbIL-15 increased the gene expression of Bcl2, STAT3, and STAT5 in cattle PBMCs. Electrophoresis of PCR products showed that rbIL-15 most actively induced the expression of Bcl2 and STAT3 genes. The STAT5 cDNA PCR product was two times less abundant compared to the PCR products of Bcl2 and STAT3. The concentration of 1 µg rIL-15 induced optimal levels of Bcl2 expression, increasing it fivefold. Recombinant IL-15 induced a tenfold increase in STAT3 expression and a ninefold increase in STAT5 expression compared to untreated cells. The role of IL-15 in inducing CPT1a, a crucial step in the transformation of activated CD8+ T cells into memory CD8+ T cells, was evaluated in vitro. The effect of recombinant IL-15 on Cpt1a expression was analyzed by quantifying the corresponding mRNA using PCR with specific primers in stimulated lymphocytes. Cpt1a, which plays an important role in CD8+ T cell memory induction, was upregulated more than twofold at 1 µg, while 500 ng resulted in a 1.5-fold increase in expression.
In parallel, culture supernatants from rbIL-15-stimulated cattle PBMCs were analyzed for IFN-γ production using ELISA. Application of 1 µg rbIL-15 per well increased IFN-γ production to 12 ng/mL. The use of 500 ng resulted in 1.92 ng/mL; 250 ng yielded 0.768 ng/mL. Untreated PBMCs produced 0.123 ng/mL of IFN-γ. Statistical comparisons between untreated and treated groups were performed using one-way ANOVA. Differences were considered statistically significant at P < 0.001.
To study the combined effect of rbIL-15 and the blockade of CTLA-4 and PD-L1 receptors using monoclonal antibodies, PBMCs from five healthy cows were used. The cells were cultured with rbIL-15 and monoclonal antibodies against bovine CTLA-4 and PD-L1, both individually and in combination. IFN-γ production in cultured PBMCs was assessed using ELISA. IFN-γ production in rbIL-15-treated cells was tenfold higher than in untreated cells. The addition of anti-CTLA-4 or anti-PD-L1 antibodies alone to rbIL-15-treated PBMCs did not lead to a statistically significant increase in IFN-γ. However, the combined use of both antibodies with rbIL-15 resulted in a statistically significant increase in IFN-γ production.
A patent application has been filed: “Microorganism strain Escherichia coli BL21/pET28/rbIL15, a producer of recombinant interleukin-15 of cattle.” Application number: 2024.0821.1
Published:
1 Mukantayev K, Tursunov K, Adish Z, Kanayev D, Tokhtarova L, Nurtleu M, and Abirbekov B (2024) Cytotoxic T-lymphocyte antigen 4 and programmed cell death ligand 1 blockade increases the effectiveness of interleukin-15 immunotherapy in a bovine leukemia model, Veterinary World, 17(9): 2096–2103. Q2. Scopus – 82 процентиль.
2 Kanatbek N. Mukantayev, Kanat A. Tursunov, Zhansaya B. Adish , Darkhan B. Kanaev, Laura A. Tokhtarova, Malika Nurtleu, Bisultan E. Abirbekov. Effects of interleukin-15 on bovine natural killer and CD8+ T cells and its potential in treating viral infections // Herald of Science of S.Seifullin Kazakh Agrotechnical Research University: Veterinary Sciences. – Astana: S. Seifullin Kazakh Аgrotechnical Research University, 2024. – № 1 (009). – P. 15-27. – ISSN 2958-5430, ISSN 2958-5449