Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells
2.2. Viruses
2.3. Infection and Neutralization of rRSV in 96-Well Cell-Culture Plates
2.4. Detection of rRSV-Fluorescent Protein Infection and Neutralization
2.5. Detection of rRSV-Luciferase Neutralization
2.6. Detection of RSV Neutralization Using ELISA
3. Results
3.1. Conditions of RSV-Fluorescent Protein Infection
3.2. Comparison of RSV Neutralization Assays Using ELISA or rRSV-Reporter Proteins
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Li, Y.; Wang, X.; Blau, D.M.; Caballero, M.T.; Feikin, D.R.; Gill, C.J.; Madhi, S.A.; Omer, S.B.; Simoes, E.A.F.; Campbell, H.; et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: A systematic analysis. Lancet 2022, 399, 2047–2064. [Google Scholar] [CrossRef] [PubMed]
- Henderson, F.W.; Collier, A.M.; Clyde, W.A., Jr.; Denny, F.W. Respiratory-syncytial-virus infections, reinfections and immunity. A prospective, longitudinal study in young children. N. Engl. J. Med. 1979, 300, 530–534. [Google Scholar] [CrossRef] [PubMed]
- Glezen, W.P.; Taber, L.H.; Frank, A.L.; Kasel, J.A. Risk of primary infection and reinfection with respiratory syncytial virus. Am. J. Dis. Child. 1986, 140, 543–546. [Google Scholar] [CrossRef] [PubMed]
- Okamoto, M.; Dapat, C.P.; Sandagon, A.M.D.; Batangan-Nacion, L.P.; Lirio, I.C.; Tamaki, R.; Saito, M.; Saito-Obata, M.; Lupisan, S.P.; Oshitani, H. Molecular Characterization of Respiratory Syncytial Virus in Children with Repeated Infections with Subgroup B in the Philippines. J. Infect. Dis. 2018, 218, 1045–1053. [Google Scholar] [CrossRef]
- Kopera, E.; Czajka, H.; Zapolnik, P.; Mazur, A. New Insights on Respiratory Syncytial Virus Prevention. Vaccines 2023, 11, 1797. [Google Scholar] [CrossRef]
- Papi, A.; Ison, M.G.; Langley, J.M.; Lee, D.G.; Leroux-Roels, I.; Martinon-Torres, F.; Schwarz, T.F.; van Zyl-Smit, R.N.; Campora, L.; Dezutter, N.; et al. Respiratory Syncytial Virus Prefusion F Protein Vaccine in Older Adults. N. Engl. J. Med. 2023, 388, 595–608. [Google Scholar] [CrossRef]
- Kampmann, B.; Madhi, S.A.; Munjal, I.; Simoes, E.A.F.; Pahud, B.A.; Llapur, C.; Baker, J.; Perez Marc, G.; Radley, D.; Shittu, E.; et al. Bivalent Prefusion F Vaccine in Pregnancy to Prevent RSV Illness in Infants. N. Engl. J. Med. 2023, 388, 1451–1464. [Google Scholar] [CrossRef]
- Walsh, E.E.; Perez Marc, G.; Zareba, A.M.; Falsey, A.R.; Jiang, Q.; Patton, M.; Polack, F.P.; Llapur, C.; Doreski, P.A.; Ilangovan, K.; et al. Efficacy and Safety of a Bivalent RSV Prefusion F Vaccine in Older Adults. N. Engl. J. Med. 2023, 388, 1465–1477. [Google Scholar] [CrossRef]
- Falsey, A.R.; Walsh, E.E. Relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults. J. Infect. Dis. 1998, 177, 463–466. [Google Scholar] [CrossRef]
- van Remmerden, Y.; Xu, F.; van Eldik, M.; Heldens, J.G.; Huisman, W.; Widjojoatmodjo, M.N. An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting. Virol. J. 2012, 9, 253. [Google Scholar] [CrossRef]
- Do, L.A.H.; Tse, R.; Nathanielsz, J.; Anderson, J.; Ong, D.S.; Chappell, K.; Mulholland, K.; Licciardi, P.V. An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay. J. Vis. Exp. 2019, 143, e59025. [Google Scholar] [CrossRef]
- Zielinska, E.; Liu, D.; Wu, H.Y.; Quiroz, J.; Rappaport, R.; Yang, D.P. Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis. Virol. J. 2005, 2, 84. [Google Scholar] [CrossRef] [PubMed]
- Varada, J.C.; Teferedegne, B.; Crim, R.L.; Mdluli, T.; Audet, S.; Peden, K.; Beeler, J.; Murata, H. A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment. Virol. J. 2013, 10, 195. [Google Scholar] [CrossRef] [PubMed]
- Boukhvalova, M.S.; Mbaye, A.; Kovtun, S.; Yim, K.C.; Konstantinova, T.; Getachew, T.; Khurana, S.; Falsey, A.R.; Blanco, J.C.G. Improving ability of RSV microneutralization assay to detect G-specific and cross-reactive neutralizing antibodies through immortalized cell line selection. Vaccine 2018, 36, 4657–4662. [Google Scholar] [CrossRef]
- Rameix-Welti, M.A.; Le Goffic, R.; Herve, P.L.; Sourimant, J.; Remot, A.; Riffault, S.; Yu, Q.; Galloux, M.; Gault, E.; Eleouet, J.F. Visualizing the replication of respiratory syncytial virus in cells and in living mice. Nat. Commun. 2014, 5, 5104. [Google Scholar] [CrossRef]
- Palsson, S.A.; Dondalska, A.; Bergenstrahle, J.; Rolfes, C.; Bjork, A.; Sedano, L.; Power, U.F.; Rameix-Welti, M.A.; Lundeberg, J.; Wahren-Herlenius, M.; et al. Single-Stranded Oligonucleotide-Mediated Inhibition of Respiratory Syncytial Virus Infection. Front. Immunol. 2020, 11, 580547. [Google Scholar] [CrossRef]
- Shambaugh, C.; Azshirvani, S.; Yu, L.; Pache, J.; Lambert, S.L.; Zuo, F.; Esser, M.T. Development of a High-Throughput Respiratory Syncytial Virus Fluorescent Focus-Based Microneutralization Assay. Clin. Vaccine Immunol. 2017, 24, e00225-17. [Google Scholar] [CrossRef]
- Sun, Y.; Lopez, C.B. Preparation of Respiratory Syncytial Virus with High or Low Content of Defective Viral Particles and Their Purification from Viral Stocks. Bio-Protocol 2016, 6, e1820. [Google Scholar] [CrossRef]
- Sun, D.; Hsu, A.; Quiroz, J.; He, X.; Whiteman, M.C.; Gurney, K.B.; Dellatore, S. Development and comparison of three cell-based potency assays for anti-respiratory syncytial virus monoclonal antibody. Biologicals 2021, 74, 1–9. [Google Scholar] [CrossRef]
- Georgakoudi, I.; Jacobson, B.C.; Müller, M.G.; Sheets, E.E.; Badizadegan, K.; Carr-Locke, D.L.; Crum, C.P.; Boone, C.W.; Dasari, R.R.; Van Dam, J.; et al. NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes. Cancer Res. 2002, 62, 682–687. [Google Scholar]
- Hosken, N.; Plikaytis, B.; Trujillo, C.; Mahmood, K.; Higgins, D. A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard. Vaccine 2017, 35, 3082–3088. [Google Scholar] [CrossRef] [PubMed]
- Raghunandan, R.; Higgins, D.; Hosken, N. RSV neutralization assays—Use in immune response assessment. Vaccine 2021, 39, 4591–4597. [Google Scholar] [CrossRef] [PubMed]
- Fix, J.; Descamps, D.; Galloux, M.; Ferret, C.; Bouguyon, E.; Zohari, S.; Naslund, K.; Hagglund, S.; Altmeyer, R.; Valarcher, J.F.; et al. Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: A proof of concept using cyclopamine. Vet. Res. 2023, 54, 36. [Google Scholar] [CrossRef] [PubMed]
- Jo, W.K.; Schadenhofer, A.; Habierski, A.; Kaiser, F.K.; Saletti, G.; Ganzenmueller, T.; Hage, E.; Haid, S.; Pietschmann, T.; Hansen, G.; et al. Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants. Proc. Natl. Acad. Sci. USA 2021, 118, e2026558118. [Google Scholar] [CrossRef]
- Takahashi, T.; Ueno, S.; Sugiura, Y.; Shimizu, K.; Kamitani, W. Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II. Microbiol. Immunol. 2023, 67, 413–421. [Google Scholar] [CrossRef]
Protocol | Plaque Counting [8,9,10] | qPCR [11] | ELISA [12] | rRSV-Cherry in This Study |
---|---|---|---|---|
RSV | EGFP-expressing rRSV [8] or RSV A2 strain [9,10] | RSV A2 or RSV B1 | RSV A2 or long or B (18537) | rRSV-Cherry |
Cell line | Vero [8,10] or A549 [9] | Vero | Hep2 or Vero or A549 | Hep2 |
Detection | Plaque counting with ELISPOT reader [8,9,10] + Immunostaining [9,10] | SYBR-green dye fluorescence | Absorbance 450 nm of HRP-substrate solution | Reporter fluorescence |
Reagents | Wash buffer, fixation buffer and antibody reagents | RNA extraction kits, SYBR-green qPCR reagents and primers for RSV N gene | Wash buffer, fixation buffer, antibody reagents and HRP-substrate solution | Wash buffer and fixation buffer |
Instruments | Plate reader specialized for ELISPOT assay | qPCR thermal cycler | Plate reader for absorbance | Plate reader for fluorescence |
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Yamagata, Y.; Toizumi, M.; Eleouet, J.-F.; Rameix-Welti, M.-A.; Takeda, M.; Yoshida, L.-M. Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein. Methods Protoc. 2025, 8, 60. https://doi.org/10.3390/mps8030060
Yamagata Y, Toizumi M, Eleouet J-F, Rameix-Welti M-A, Takeda M, Yoshida L-M. Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein. Methods and Protocols. 2025; 8(3):60. https://doi.org/10.3390/mps8030060
Chicago/Turabian StyleYamagata, Yutaro, Michiko Toizumi, Jean-Francois Eleouet, Marie-Anne Rameix-Welti, Makoto Takeda, and Lay-Myint Yoshida. 2025. "Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein" Methods and Protocols 8, no. 3: 60. https://doi.org/10.3390/mps8030060
APA StyleYamagata, Y., Toizumi, M., Eleouet, J.-F., Rameix-Welti, M.-A., Takeda, M., & Yoshida, L.-M. (2025). Improved RSV Neutralization Assay Using Recombinant RSV Expressing Reporter Fluorescent Protein. Methods and Protocols, 8(3), 60. https://doi.org/10.3390/mps8030060