A Novel Immunofluorescent Test System for SARS-CoV-2 Detection in Infected Cells
Alexandra Cancer, Victoria Matyushenko, Polina Prokopenko, Arina Kostromitina, Dmitry Polyakov, Alexey Sokolov, Larisa Rudenko, Irina Isakova-Sivak
Abstract
Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients. However, some aspects of the assay, such as sensitivity and time cost, need improvement. Here, we developed an express test, which may be potentially used in clinical practice for the assessment of serum-caused SARS-CoV-2 inhibition in infected cell cultures. It implies the detection and counting of coronaviral fluorescent-forming units (FFU) and includes two sequentially used developing components: biotinylated mouse monoclonal antibodies against the recombinant N protein of SARS-CoV-2 (B.1) and the recombinant EGFP-streptavidin fusion protein. Due to the universal specificity of the antibodies, our analytical tool is suitable for the detection of various strains of SARS-CoV-2 when determining both the infectious titer of viruses and the titer of serum virus-neutralizing antibodies. The developed two-component test system is characterized by high sensitivity, a reduced number of analytic stages and low assay cost, as well as by flexibility, since it may be modified for detection of other pathogens using the appropriate antibodies.
Introduction
COVID-19 is an acute respiratory infection caused by the coronavirus SARS-CoV-2, characterized by high contagiousness and mortality of the infected population, which determines its high socio-economic significance. The global COVID-19 pandemic, which began in late 2019, has affected more than 704 million people and took the lives of 7 million victims [1]. Despite to date the pandemic is finished, the disease cases continue to emerge in the form of successive waves of the worldwide spread of new SARS-CoV-2variants, which is associated with the high antigenic variability of the virus [2]. The rapid and precise COVID-19 diagnostics are required to control the spread of infection and to assess the population immunity. There are several widely used methods of express COVID-19 testing, such as PCR, the detection of viral antigens, or serological measurement of antiviral antibody titer by ELISA, LFIA, CLIA or microneutralization assay (MN) assay [3]. Despite the fact that MN reaction, as one of the serological approaches, is not applicable to reveal the acute phase of COVID-19, it still considered to be “the gold standard” to assess both the intensity of the formation of antiviral antibodies and their functional activity, that is, the ability to abolish the viral propagation [4]. These data are important not only for individual COVID-19 diagnostics, but also for assessment of the rate of infection spread and herd immunity state [5].
Materials and methods
2.1. Cells, viruses and monoclonal antibodies
The following SARS-CoV-2 viruses belonging to different lineages were obtained from the Smorodintsev Research Institute of Influenza (Saint Petersburg, Russia):
African green monkey kidney Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and routinely maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic–antimycotic (AA) (all from Capricorn Scientific, Ebsdorfergrund, Germany).
The viruses were propagated on the Vero E6 cells using DMEM supplemented with 2% FBS, 10 mM of HEPES and 1× antibiotic-antimycotic (all from Capricorn Scientific, Ebsdorfergrund, Germany) at 37°C and 5% CO2. All experiments with live SARS-CoV-2 were performed in a biosafety-level-3 laboratory (BSL-3).
NCL5 antibodies against the N protein of SARS-CoV-2 (B.1), specifically binding N proteins of B.1.351, P.1, B.1.617.2 and B.1.1.529 VOCs were previously obtained [11] using standard hybridoma approach, isotyped as IgG2a, dialyzed against 20 mM phosphate buffer, pH 7.4 (PBS), aliquoted and stored at -20°C.
Results
3.1. EGFP-SA obtaining
The recombinant fusion protein EGFP-SA was expressed in IPTG-induced transformants of E.coli strain BL21(DE3) and purified from the biomass lysate using IMAC. The model of EGFP-SA based on the known X-ray structures of the components of the fusion protein is shown in Fig 1A. SDS-PAGE analysis confirmed the predicted molecular mass of EGFP-SA (about 42 kDa) as well as purity of the obtained protein (Fig 1C). Furthermore, assessment of fusion protein stability at different storage conditions revealed it instability after the thaw-freezing cycles, suggesting that functionality of the protein can only be maintained when stored at +4°C (Fig 1D–1F).
Discussion
The neutralizing antibodies are detected in approximately 40%-70% of SARS-CoV-2-infected individuals, correlate with COVID-19 severity and are reported as key players in preventing viral entry into the host cell, so MN titer may be considered as an indicator of antiviral protection [18–20]. It is well known that serum IgG antibody levels, similarly with the serum neutralizing activity gradually decrease in COVID-19 convalescents, but don’t disappear completely for a long time [21,22]. The maintenance of virus-neutralizing antibodies after SARS-CoV-2 infection was observed for up to several months after infection [18] and they may be boosted by revaccination. Thus, although MN titer measurement is not an appropriate method for acute COVID-19 testing, it seems to be suitable for evaluation of the humoral immunity persistence, especially in a context of possible revaccination requirements. Several protocols have previously been proposed for performing MN assays to test SARS-CoV-2 inhibition by purified antibodies or serum [4,23–28]. The most widely used approaches to detect the virus in infected cells are non-specific assessment of virus titer by CPE and ELISAs implying antiviral monoclonal antibodies. Interestingly, Bennett et al. used the secondary antibody labeled with Alexa594 to detect SARS-CoV-2 and automatically count the percentage of infected cells [29]. In order to develop the test system with universal strain specificity, we used cross-strain N-specific biotinylated antibody and fluorescent developing fusion protein for the first time and reported the preliminary data on serum neutralizing activity in SARS-CoV-2 naïve donors, Sputnik V-vaccinated individuals and COVID-19 convalescents up to 16 months post-symptom onset, while the longevity of these responses will be further studied.
Conclusion
The proposed assay of SARS-CoV-2 detection in infected cell cultures may be successfully used to rapidly evaluate the serum MN titers in patients who have recovered from COVID-19. The described method seems to be a powerful tool to assess the levels of virus-neutralizing antibodies generated upon natural infection or administration of novel COVID-19 vaccine candidates. In contrast to traditional Spike-targeting tests designed to evaluate the humoral immune responses induced by infection or vaccination, our approach allows to estimate the capacity of vaccines to establish the cross-strain B-cell responses, which indicates its protective potential. Notably, this study used evolutionarily distant SARS-CoV-2 strains obtained upon different waves of COVID-19; therefore, given the advantages obtained, the developed FFU-targeting test system can be recommended for universal assessment of individual viral neutralizing antibody titers regardless of the SARS-CoV-2 strain that caused COVID-19.
Acknowledgments
We thank Dr. Ekaterina Stepanova for her advices on SARS-CoV-2 propagation and technical assistance, as well as all blood donors who participated in the study.
Citation: Rak A, Matyushenko V, Prokopenko P, Kostromitina A, Polyakov D, Sokolov A, et al. (2024) A novel immunofluorescent test system for SARS-CoV-2 detection in infected cells. PLoS ONE 19(5): e0304534. https://doi.org/10.1371/journal.pone.0304534
Editor: Boyan Grigorov, CRCL: Cancer Research Center of Lyon, FRANCE
Received: February 26, 2024; Accepted: May 14, 2024; Published: May 31, 2024
Copyright: © 2024 Rak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The minimal data cannot be shared publicly because of its confidentiality. The dataset used to prepare the article represents optical density and FFU values in the plate wells, so uploading them to public repositories without descriptions of groups, samples and dilutions (including location of samples within the plates) seems to be inappropriate. We uploaded a raw dataset related to our manuscript to the repository of the Local Ethics Committee of the Institute of Experimental Medicine (email: iemetcom@yandex.ru), located on Yandex Disk at: https://disk.yandex.ru/client/disk/Datasets%20for%20External%20Requests/Rak%20et%20al.%202024%20PLoS%20One%20(FFU%20test%20system)%20raw%20data%20set Access to this repository is provided by the Local Ethics Committee staff using the login iemetcom@yandex.ru, and data may be stored there indefinitely. The minimal data are available for researchers who meet the criteria for access to confidential data upon request to the following member of the IEM Local Ethics Committee who is responsible for ensuring data access: Dr. Olga V. Kirik, email: olga_kirik@mail.ru, mob: +7 (951) 654-94-23.
Funding: This research was funded by the RSCF grant 21-75-30003. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0304534#abstract0
