Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

Genova, Cecilia Di; Sampson, Alex; Scott, Simon D.; Cantoni, Diego; Mayora-Neto, Martin; Bentley, Emma; Mattiuzzo, Giada; Wright, Edward; Derveni, Mariliza; Auld, Bethany; Ferrara, Bill T; Harrison, Dale J A; Said, Mohamed; Selim, Arwa; Thompson, Edward P.; Thompson, Craig; Carnell, George; Temperton, Nigel

doi:10.21769/bioprotoc.4236

Cited by 47 publications

(43 citation statements)

References 23 publications

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“…We generated pseudotyped viruses (PVs) bearing the Spike protein of the SARS-CoV-2 Wuhan Type and VOCs as previously described ( 35 ). Briefly 1000ng of p8.91 HIV Gag-pol, 1500ng of pCSFLW luciferase and 1000ng of SARS-CoV-2 Spike plasmids were resuspended in Opti-MEM and mixed with FuGENE HD (Promega) at a 1:3 ratio.…”

Section: Methodsmentioning

confidence: 99%

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Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds

et al. 2022

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The rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage. Using pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL. Our data demonstrate a significant reduction in the ability of first wave convalescent sera to neutralise the VOCs. Patients and HCWs with more severe COVID-19 were found to have higher antibody titres and to neutralise the VOCs more effectively than individuals with milder symptoms. Using an estimated threshold for 50% protection, 54 IU/mL, we found most asymptomatic and mild cases did not produce titres above this threshold.

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Section: Methodsmentioning

confidence: 99%

“…Pseudotype microneutralisation assays (pMN) were carried out as previously described ( 36 ). Briefly, human convalescent serum was mixed with DMEM at a 1:40 input dilution and serially diluted 2-fold to 1:5,120 in a white F-bottom 96 well plate.…”

Section: Methodsmentioning

confidence: 99%

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds

et al. 2022

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“…PVs were harvested 48 hours post-transfection by filtering culture media through a 0.45 µm cellulose acetate filter. PVs were then titrated and aliquoted for storage at −70°C 40 .…”

Section: Methodsmentioning

confidence: 99%

“…Luminescence was measured using a GloMax luminometer (Promega). PVs were quantified based on relative luminescence units per ml (RLU/ml) 40 .…”

Section: Methodsmentioning

confidence: 99%

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients

et al. 2022

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RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

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“…The diluted sera (50µL) were mixed with pseudotyped SARS-CoV-2 viruses (650 TCID 50 per well) in 96-well plates and incubated at 37°C and 5% CO 2 for 1 h. Then the 293T-ACE2-TMPRSS2 cells were added and co-cultured for the next 24h. 14 The chemiluminescence signals were measured in relative luminescence units (RLU) using a Bright GloTM luciferase assay system with a GloMax® Navigator Microplate Luminometer. The neutralizing titers (NAT50) were defined as the 50% inhibitory dilution (ID50) which was calculated with the highest dilution of plasma that resulted in a 50% reduction of relative light units compared with virus control.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive humoral and cellular immune responses to SARS-CoV-2 variants in diverse Chinese populations: A benefit perspective of national vaccination

Long

et al. 2022

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The emerging SARS-CoV-2 variants have made great challenges to current vaccine and pandemic control strategies. B.1.1.529 (Omicron), which was classified as a variant of concern (VOC) by the World Health Organization on November 26th, 2021, has quickly become the dominant circulating variant and causing waves of infections. It is urgent to understand the current immune status of the general population given that pre-existing immunity has been established by national vaccination or exposure to past variants. Using sera from 85 individuals (including 21 convalescents of natural infection, 15 cases suffered a breakthrough infection after vaccination, and 49 vaccinated participants without infection history), we showed that the cross-neutralizing activity against VOCs such as Omicron can be detected in 53 (62.4%) cases, although less potent than against the Wuhan-1 strain (WT), with a 3.9-fold reduction in geometric mean neutralizing titer (GMT) (130.7, 95% CI 88.4-193.3 vs 506, 355.8-719.7, respectively). Subgroup analysis revealed significantly enhanced neutralizing activity against WT and VOCs in Delta convalescent sera. The neutralizing antibodies against Omicron were detectable in 75% of convalescents and 44.9% of healthy donors (p = 0.006), with a GMT of 289.5, 180.9-463.3 and 42.6, 31.3-59, respectively. However, the protective effect against VOCs was weaker in young convalescents (aged < 18y), with a detectable rate of 50% and a GMT of 46.4 against Omicron, similar to vaccinees. The pan-sarbecovirus neutralizing activities were not observed in vaccinated SARS-CoV-1 survivors. A booster dose significantly increased the breadth and magnitude of neutralization against WT and VOCs to different degrees than full vaccination. In addition, we showed that COVID-19 inactivated vaccines can elicit Omicron-specific T cell responses. The positive rates of ELISpot reactions were 26.7% (4/15) and 43.8% (7/16) in the full vaccination group and the booster vaccination group, respectively. The neutralizing antibody titers declined while T-cell responses remain robust over 6 months. These findings will inform the optimization of public health vaccination and intervention strategies to protect diverse populations against SARS-CoV-2 variants.

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Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

Cited by 47 publications

References 23 publications

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients

Comprehensive humoral and cellular immune responses to SARS-CoV-2 variants in diverse Chinese populations: A benefit perspective of national vaccination

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