Proper Selection of In Vitro Cell Model Affects the Characterization of the Neutralizing Antibody Response against SARS-CoV-2

Criscuolo, Elena; Giuliani, Benedetta; Ferrarese, Roberto; Diotti, Roberta Antonia; Clementi, Massimo; Mancini, Nicasio; Clementi, Nicola

doi:10.3390/v14061232

Cited by 3 publications

(5 citation statements)

References 38 publications

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“…S1, Supplementary Material online). These results corroborated well with previous published data ( Ren et al 2006 ; Aguiar et al 2020 ; Matsuyama et al 2020 ; Koch et al 2021 ; Criscuolo et al 2022 ; Zeng et al 2022 ).…”

Section: Resultssupporting

confidence: 93%

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells

Aiewsakun

Phumiphanjarphak

Ludowyke

et al. 2023

Genome Biology and Evolution

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, and scientists around the world are currently studying the virus intensively in order to fight against the on-going pandemic of the virus. To do so, SARS-CoV-2 is typically grown in the lab to generate viral stocks for various kinds of experimental investigations. However, accumulating evidence suggests that such viruses often undergo cell culture adaptation. Here, we systematically explored cell culture adaptation of two SARS-CoV-2 variants, namely the B.1.36.16 variant and the AY.30 variant, a sub lineage of the B.1.617.2 (Delta) variant, propagated in three different cell lines, including Vero E6, Vero E6/TMPRSS2, and Calu-3 cells. Our analyses detected numerous potential cell culture adaptation changes scattering across the entire virus genome, many of which could be found in naturally circulating isolates. Notable ones included mutations around the spike glycoprotein's multibasic cleavage site, and the Omicron-defining H655Y mutation on the spike glycoprotein, as well as mutations in the nucleocapsid protein's linker region, all of which were found to be Vero E6-specific. Our analyses also identified deletion mutations on the non-structural protein 1 and membrane glycoprotein as potential Calu-3-specific adaptation changes. S848C mutation on the non-structural protein 3, located to the protein's papain-like protease domain, was also identified as a potential adaptation change, found in viruses propagated in all three cell lines. Our results highlight SARS-CoV-2 high adaptability, emphasize the need to deep-sequence cultured viral samples when used in intricate and sensitive biological experiments, and illustrate the power of experimental evolutionary study in shedding lights on the virus evolutionary landscape.

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Section: Resultssupporting

confidence: 93%

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells

Aiewsakun

Phumiphanjarphak

Ludowyke

et al. 2023

Genome Biology and Evolution

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“…Cell viability was assessed using the Cell Proliferation kit II (XTT) (Roche Diagnostics, Merck, Darmstadt, Germany) as previously described [ 59 ]. In this assay, the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) was cleaved by viable cells in order to form an orange formazan dye that is then quantified photometrically at 450 nm.…”

Section: Methodsmentioning

confidence: 99%

“…A clinical isolate of SARS-CoV-2 Omicron BA.5 (hCoV-19/Italy/LOM-UniSR38/2022, GISAID ID: EPI_ISL_15778241) was obtained and propagated in Vero E6 cells, and the viral titer was determined by the 50% tissue culture infective dose (TCID 50 ) and plaque assay. All the infection experiments were performed in a biosafety level three (BSL-3) laboratory, as previously described [ 59 ].…”

Section: Methodsmentioning

confidence: 99%

“…Virus stocks were titrated using the Endpoint Dilutions Assay (EDA, TCID 50 /mL), as described [ 59 ]. Briefly, Vero E6 cells (4 × 10 5 cells/mL) were seeded into 96 wells plates and infected with base 10 dilutions of the virus stock.…”

Section: Methodsmentioning

confidence: 99%

“…After a PBS wash to remove cell-free virus particles, 3CLpro-inhibitor-containing medium with 2% FBS was added to the cells and was maintained until the end of the experiment [48 h post-infection (hpi) for F-CoV (CRFK), 72 hpi for SARS-CoV-2 (Vero E6), and 96 hpi for B-CoV (HRT-18) and HCoV-OC43 (HRT-18)]. Then, the SARS-CoV-2 cytopathic effect (CPE) was assessed using a scoring system (0 = uninfected; 0.5 to 2.5 = increasing number/area of plaques; 3 = all the cells are infected), as described [ 59 ]. The infection control (score 3) was set as 100% infection and the uninfected cells (score 0) were set as 0% infection.…”

Section: Methodsmentioning

confidence: 99%

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Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity

et al. 2023

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The SARS-CoV-2 protease (3CLpro) is one of the key targets for the development of efficacious drugs for COVID-19 treatment due to its essential role in the life cycle of the virus and exhibits high conservation among coronaviruses. Recent studies have shown that flavonoids, which are small natural molecules, have antiviral activity against coronaviruses (CoVs), including SARS-CoV-2. In this study, we identified the docking sites and binding affinity of several natural compounds, similar to flavonoids, and investigated their inhibitory activity towards 3CLpro enzymatic activity. The selected compounds were then tested in vitro for their cytotoxicity, for antiviral activity against SARS-CoV-2, and the replication of other coronaviruses in different cell lines. Our results showed that Baicalein (100 mg/mL) exerted strong 3CLpro activity inhibition (>90%), whereas Hispidulin and Morin displayed partial inhibition. Moreover, Baicalein, up to 25 mg/mL, hindered >50% of SARS-CoV-2 replication in Vero E6 cultures. Lastly, Baicalein displayed antiviral activity against alphacoronavirus (Feline-CoV) and betacoronavirus (Bovine-CoV and HCoV-OC43) in the cell lines. Our study confirmed the antiviral activity of Baicalein against SARS-CoV-2 and demonstrated clear evidence of its pan-coronaviral activity.

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Infection kinetics, syncytia formation, and inflammatory biomarkers as predictive indicators for the pathogenicity of SARS-CoV-2 Variants of Concern in Calu-3 cells

Purwono,

Vacharathit,

Manopwisedjaroen

et al. 2024

PLoS ONE

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The ongoing COVID-19 pandemic has led to the emergence of new SARS-CoV-2 variants as a result of continued host-virus interaction and viral genome mutations. These variants have been associated with varying levels of transmissibility and disease severity. We investigated the phenotypic profiles of six SARS-CoV-2 variants (WT, D614G, Alpha, Beta, Delta, and Omicron) in Calu-3 cells, a human lung epithelial cell line. In our model demonstrated that all variants, except for Omicron, had higher efficiency in virus entry compared to the wild-type. The Delta variant had the greatest phenotypic advantage in terms of early infection kinetics and marked syncytia formation, which could facilitate cell-to-cell spreading, while the Omicron variant displayed slower replication and fewer syncytia formation. We also identified the Delta variant as the strongest inducer of inflammatory biomarkers, including pro-inflammatory cytokines/chemokines (IP-10/CXCL10, TNF-α, and IL-6), anti-inflammatory cytokine (IL-1RA), and growth factors (FGF-2 and VEGF-A), while these inflammatory mediators were not significantly elevated with Omicron infection. These findings are consistent with the observations that there was a generally more pronounced inflammatory response and angiogenesis activity within the lungs of COVID-19 patients as well as more severe symptoms and higher mortality rate during the Delta wave, as compared to less severe symptoms and lower mortality observed during the current Omicron wave in Thailand. Our findings suggest that early infectivity kinetics, enhanced syncytia formation, and specific inflammatory mediator production may serve as predictive indicators for the virulence potential of future SARS-CoV-2 variants.

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Proper Selection of In Vitro Cell Model Affects the Characterization of the Neutralizing Antibody Response against SARS-CoV-2

Cited by 3 publications

References 38 publications

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells

Natural Flavonoid Derivatives Have Pan-Coronavirus Antiviral Activity

Infection kinetics, syncytia formation, and inflammatory biomarkers as predictive indicators for the pathogenicity of SARS-CoV-2 Variants of Concern in Calu-3 cells

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