PREPARATION OF MONOCLONAL ANTIBODIES FOR DETECTION OF RECOMBINANT FLAGELLIN C FROM PSEUDOMONAS AERUGINOSA
- Authors: Zherebtsov A.P.1, Yakovleva I.V.2, Gavrilova N.F.2, Mikhailova N.A.2
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Affiliations:
- I. Mechnikov Research Institute of Vaccines and Sera
- Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov."
- Section: Joint Immunology Forum 2024
- URL: https://rusimmun.ru/jour/article/view/16865
- DOI: https://doi.org/10.46235/1028-7221-16865-POM
- ID: 16865
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Abstract
An important virulence factor in the pathogenesis of infections caused by Pseudomonas aeruginosa is flagellin: it serves as the main structural component of the bacterial flagellum and an acceptor for the TLR5 receptor of the innate immune system. Toll-like receptor 5 is able to bind bacterial flagellin and activate the anti-inflammatory transcription factor NF-kB through the adapter protein MyD88, which induces the production of anti-inflammatory cytokines. The inclusion of flagellin in recombinant proteins increased the ability to stimulate the production of anti-inflammatory cytokines and activate antigen-presenting cells. A number of experiments have shown that the use of flagellin as a molecular adjuvant in vaccines increases the expression of CD80, CD83, CD86 and MHCII molecules on the surface of dendritic cells, and also leads to an increase in the secretion of IFN-γ and α-defensins by dendritic and NK cells; T cell proliferation and activation of antigen-specific cytotoxic T lymphocytes, as well as increased induction of antigen-specific IgG and IgA antibodies.
Due to the natural and acquired resistance of P. aeruginosa to antibiotics, the available choice of antipseudomonas drugs is decreasing, and therefore the problem of developing effective therapeutic drugs to protect against this infection is of high medical and social importance. For this purpose, it seems promising to study the immunobiological properties of P. aeruginosa flagellin as a possible vaccine component.
Based on this, in the laboratory of protective antigens of the Federal State Budgetary Institution NIIVS named after. I.I. Mechnikov, recombinant flagellin C (FliC) of P. aeruginosa was obtained, its immunogenicity and protective properties were proven. However, the question of standardizing methods for screening and monitoring the resulting recombinant FliC protein remains open. To solve this issue, hybridomas producing monoclonal antibodies (mAb) of a given specificity were obtained, the basic immunochemical properties of mAbs were studied, and the possibility of using them as reagents in constructing a test system for identifying and standardizing the recombinant FliC protein upon its production was assessed.
Purpose of the work: To obtain monoclonal antibodies to the recombinant flagellin C protein of P. aeruginosa, to study their basic immunochemical properties and to evaluate the possibility of using the recombinant FliC protein for screening and control.
About the authors
Anton Pavlovich Zherebtsov
I. Mechnikov Research Institute of Vaccines and Sera
Author for correspondence.
Email: antonzherebtsov@yandex.ru
ORCID iD: 0009-0008-1676-9515
Researcher at the Laboratory of Protective Antigens.
Russian Federation, Maly Kazenny Lane, 5A, Moscow, 105064, Russian FederationIrina Vladimirovna Yakovleva
Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov."
Email: dissovet-mech@mail.ru
ORCID iD: 0000-0001-5123-2651
PhD, Leading Researcher at the Laboratory of Therapeutic Vaccines
Russian Federation, Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov." Maly Kazenny Lane, 5A, Moscow, 105064, Russian FederationNatalya Fyodorovna Gavrilova
Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov."
Email: beresina54@mail.ru
ORCID iD: 0000-0001-6704-0339
PhD, Senior Researcher, Laboratory of Therapeutic Vaccines
Russian Federation, Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov." Maly Kazenny Lane, 5A, Moscow, 105064, Russian FederationNatalya Alexandrovna Mikhailova
Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov."
Email: n_michailova@inbox.ru
ORCID iD: 0000-0002-6652-2093
Dr. Sci. (Med.). prof. head lab. protective antigens of the Federal State Budgetary Scientific Institution NIIVS named after I.I. Mechnikova
Russian Federation, Federal State Budgetary Scientific Institution “Research Institute of Vaccines and Serums named after. I.I. Mechnikov." Maly Kazenny Lane, 5A, Moscow, 105064, Russian FederationReferences
- Cui B, Liu X, Fang Y, Zhou P, Zhang Y, Wang Y. Flagellin as a vaccine adjuvant. Expert Rev Vaccines. 2018;17(4):335–49. https://doi.org/10.1080/14760584.2018.1457443
- Spagnolo A. M., Sartini M., Cristina M. L. (2021). Pseudomonas aeruginosa in the healthcare facility setting. Rev. Med. Microbiol. 32 (3), 169–175. doi: 10.1097/MRM.0000000000000271 [CrossRef] [Google Scholar]
- Chiang H.-Y., Chen T.-C., Lin C.-C., Ho L.-C., Kuo C.-C., Chi C.-Y. (2021). Trend and predictors of short-term mortality of adult bacteremia at emergency departments: a 14-year cohort study of 14 625 patients. Open Forum Infect. Dis. 8 (11). doi: 10.1093/ofid/ofab485 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Hajam IA, Dar PA, Shahnawaz I, Jaume JC, Lee JH. Bacterial flagellin-a potent immunomodulatory agent. Exp Mol Med. 2017 Sep 1; 49 (9):e373. doi: 10.1038/emm.2017.172.
- Dukhovlinov IV, Bogomolova EG, Fedorova EA. Protective activity study of a candidate vaccine against rotavirus infection based on recombinant protein FliCVP6VP8. Medical Immunology. 2016;18 (5):417–24 (In Russ.).
- Cui B, Liu X, Fang Y, Zhou P, Zhang Y, Wang Y. Flagellin as a vaccine adjuvant. Expert Rev Vaccines. 2018. Vol. 17(4). P. 335-349. doi: 10.1080/14760584.2018.1457443.
- Safronov GA, Murzina EV, Bolekhan VN, Veselova OM, Simbirtsev AS. Perspective direction of using flagellin-based drugs. Medical Academic Journal. 2017; 17(2):7–20 (In Russ.). EDN: WUERXU
- Felgner S, Spöring I. The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy. Int J Cancer. 2020. Vol. 15; 147 (2). P. 448-460. doi: 10.1002/ijc.32807.
- Burchanov Y.I. Monoclonal antibodies: from creation to clinical use // Clinical oncohematology, fundamental research and clinical practice. – 2016. – T. 9, No. 3. - P. 237-244.
- Barnea Y, Carmeli Y, Neville LF, Kahel-Reifer H, Eren R, Dagan S, Navon-Venezia S. Therapy with anti-flagellin A monoclonal antibody limits Pseudomonas aeruginosa invasiveness in a mouse burn wound sepsis model. Burns. 2009 May; 35 (3):390-6. doi: 10.1016/j.burns.2008.08.014. Epub 2008 Oct 31. PMID: 18951715.
- Zherebtsov A.P., Kaloshin A.A., Mikhailova N.A. Production and immunological characterisation of recombinant flagellin C of Pseudomonas aeruginosa. Biological Products. Prevention, Diagnosis, Treatment. 2024; 24 (1):91-102. https://doi.org/10.30895/2221-996X-2024-24-1-91-102
- Kaloshin AA, Zherebtsov AP, Mikhailova NA. Recombinant plasmid DNA pPA-FlicC encoding synthesis of recombinant flagellin-C Pseudomonas aeruginosa, strain Escherichia coli PA-FlicC – producer of the hybrid recombinant protein and a method for producing said protein. Patent of the Russian Federation No. № 2793753; 2023 (In Russ.). EDN: RAOQAM.
- Avdeeva L.V, Aleynikova T.L, Andrianova L.E., et al. Physico-chemical properties of proteins and methods of their isolation // Biochemistry textbook. – 2019. - No. 5. – pp. 66-72.
- Laemmli U.K. Cleavage of structural proteins during the assembly of head of bacteriophage T4 / U.K. Laemmli // Nature. – 1970. – V.227. – P. 680-685.