The main measures for preventing and recovering farms from brucellosis and bovine leukemia are the diagnosis and slaughter of infected animals. Despite ongoing veterinary activities, the widespread occurrence of disease has increased interest in studying immune checkpoints for the treatment of chronic infections in cows. It was determined that the progression of bovine leukemia virus (BLV) increases the concentration of regulatory T lymphocytes, leading to increased production of transforming growth factor-β (TGF-β). Increased TGF-β, in turn, suppresses the expression of interferon-γ (IFN-γ), tumor necrosis factor (TNF-α) and suppresses natural killer (NK) cells. An association between the PD-1/PD-L1 receptor and lymphocyte activation gene 3 (LAG-3) signaling pathways and BLV infection has been noted. In addition, increased expression of CTLA-4 in T cells has been reported with the progression of BLV infection. T lymphocytes, as well as the production of the cytokines IFN-γ and TNF-α, play an essential role in creating immunity against bacterial infections by increasing the activation of macrophages and dendritic cells. However, in late subclinical stages, T-cell activity decreases, allowing bacterial growth and progression to clinical disease.
The project’s novelty lies in blocking two control points to enhance immunity against the viral and bacterial load on the body of animals during chronic infections. Recent studies have shown that CTLA-4 is upregulated in chronic viral infections and malignancies, contributing to host immune dysfunction. On the other hand, the blockade of CTLA-4 binding to CD80 or CD86 by antibodies restores the immune response against these diseases. In a number of studies where the authors used recombinant bovine CTLA-4-Ig, the immune inhibitory function of CTLA-4 in cattle was proven. Immunization of mice with recombinant bovine CTLA4-Ig induced the formation of anti-CTLA-4 antibodies. Anti-CTLA-4 antibody significantly increased IFN-γ production by immune cells in healthy and BLV-infected cattle. According to the authors, antibodies against CTLA-4 may be helpful in the development of new therapy against BLV infection.
A similar effect was observed with the blockade of the PD-1/PD-L1 pathway, which enhanced T cell function and inhibited BLV proliferation. Decreasing the concentration of PD-1+ T cells through their binding to PD-L1 on B cells has been shown to contribute to the progression of viral infection. Treatment of HIV-infected macaques and LCMV-infected mice with antibodies to PD-L1 or PD-1 restored multiple functions of previously exhausted T cells and resulted in viral clearance in vivo. According to the authors, blocking the PD-1/PD-L1 pathway has potential clinical applications for enhancing host antimicrobial immunity to treat chronic infections.
To increase the activity of cows’ immunity against BLV and brucellosis by blocking CTLA-4 and PD-L1 receptors using monoclonal antibodies. Obtain strains producing recombinant extracellular domains of CTLA-4 and PD-L1 receptors of bovine immune system cells and specific monoclonal antibodies.
As a result of the research, two strains of microorganisms producing recombinant bovine CTLA-4 and PD-L1 proteins will be obtained. The conditions for the isolation and purification of recombinant proteins will be worked out and their immunobiological properties will be determined. A library of hybridoma cell lines producing monoclonal antibodies against bovine CTLA-4 and PD-L1 receptors will be obtained. The productive characteristics of hybridoma cell lines and the immunochemical properties of monoclonal antibodies will be determined. Methods will be developed to increase the activity of the immune system of cows during chronic viral and bacterial infections. The synergistic effect of monoclonal antibody therapy in the treatment of animals infected with BLV will be determined. The doses of monoclonal antibodies against bovine CTLA-4 and PD-L1 receptors will be established.
Borovikov Sergey Nikolaevich. Candidate of Biological Sciences, Associate Professor. h-index – 2, Researcher ID: AAE-7841-2022; ORCID:0000-0002-9721-9732; Author ID Scopus: 56058619600
Mukantayev Kanatbek Naizabekovich. Doctor of Biological Sciences, Associate Professor. Scopus h-index – 4, Researcher ID: AAE-7841-2022; ORCID:0000-0002-9721-9732; Author ID Scopus: 56058619600.
Tursunov Kanat Akhmetovich, PhD, Scopus h-index 3. Author ID Scopus – 57193579180, Researcher ID Web of Science N-6319-2017.
Adish Zhansaya Batyrbekkyzy, PhD doctoral student in biology, h-index 2. Author ID Scopus – 57202535857.
Kanayev Darkhan Babanovich, Master of Biological Sciences, h-index 1. Researcher ID Web of Science N-6950-2017.
Tokhtarova Laura. Junior Researcher. ORCID:0000-0003-4386-993X.
Nurtleu Malika. Junior Researcher. Scopus Author ID: 57202536508; Researcher ID: N-6297-2017; ORCID: 0000-000-1299-8782.
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2 Bulashev A.K., Borovikov S.N., Serikova S.S., Suranshiev Z.A., Kiyan V.S., Eskendirova S.Z. 2016: Development of an ELISA using anti-idiotypic antibody for diagnosis of opisthorchiasis. Folia Parasitol. 63: 025; DOI:10.14411/fp.2016.025; Web of Science Q3.
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5 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 // Iranian Journal of Veterinary Research. – 2017. –Vol.18(1). P.36-42. PMID: 28588631. Percentile – 62. Citation – 4.
6 Dmitriy V Sotnikov, Anna N Berlina, Anatoly V Zherdev, Saule Z Eskendirova, Kassym K Mukanov, Yerlan M Ramankulov, Kanatbek N Mukantayev and Boris B Dzantiev. Immuno-chromatographic Serodiagnosis of Brucellosis in Cattle Using Gold Nanoparticles and Quantum Dots // International Journal of Veterinary Science, 2019, 8(1): 28-34. DOI: 10.3390/app10030738. Percentile – 33. Citation – 5.
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9 Adish Zhansaya,Mukantayev Kanatbek, Tursunov Kanat, Ingirbay Bakhytkali, Kanayev Darkhan, Kulyyassov Arman, Tarlykov Pavel, Mukanov Kasym, Ramankulov Yerlan. Recombinant Expression and Purification of Extracellular Domain of the Programmed Cell Death Protein Receptor // Reports of Biochemistry & Molecular Biology.Vol.8, No.4, Jan 2020. http://rbmb.net/article-1-391-en.pdf3.
Based on the pET28 expression vector, two genetic constructs containing the genes for the extracellular domains of the bovine PD-1 and CTLA-4 receptors were designed. The genes for the extracellular domains of the bovine PD-1 and CTLA-4 receptors were synthesized de novo with a length of 600 and 400 base pairs, respectively. Sequencing of the synthesized genes showed a 100% match with the predicted nucleotide sequence. The synthesized genes were cloned into the expression plasmid pET28 and produced in a concentration of 208 – 328 ng/μl, a volume of 200 μl.
As a result of the transformation of E. coli strain BL21 with genetic constructs pET28/PD-1 and pET28/CTLA-4, producer strains E. coli BI21/pET28/PD-1 and E. coli BL21/pET28/CTLA-4 were obtained. It was established that the resulting cell strains stably produced recombinant proteins. The molecular weights of the proteins were 21 and 34 kDa, corresponding to the predicted mass of the desired proteins. Purified preparations of recombinant bovine CTLA-4 and PD-L1 proteins were obtained.
As a result of hybridization of B-lymphocytes from mice immunized with rCTLA-4 protein with myeloma cells X63-Ag8.653, growth of hybrid cell clones was noted in 100 wells out of 576. From the resulting hybrid cells, 8 clones produced antibodies against rCTLA-4 protein. When B-lymphocytes from mice immunized with rPD-L1 protein were hybridized with X63-Ag8.653 myeloma cells, growth of hybrid cell clones was observed in 127 out of 576 wells. Of the resulting clones of hybrid cells, 14 clones produced antibodies against rPD-L1 protein. To obtain stable producers of monoclonal antibodies, the resulting clones were cloned 3 times using the limiting dilution method. The most promising clones 1c3 and 4h8, producing mAb against bovine rCTLA-4, and clone 3g2, producing mAb against bovine rPD-L1, were selected for further work. The binding constant of mAb (Kaff (M-1)) for 1c3 was 2.9 × 108M-1; 4h8 – 2.7×108M-1 and 3g2 – 6.4×108M-1.