Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Kalemera, Mphatso D; Capella-Pujol, Joan; Chumbe, Ana; Underwood, Alexander; Bull, Rowena A.; Schinkel, Janke; Sliepen, Kwinten; Grove, Joe

doi:10.1101/2020.06.18.159442

Cited by 6 publications

(6 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All cells were cultured at 37 C and 5% CO 2 in growth medium containing high glucose Dulbecco's Modified Eagle Medium (ThermoFisher Scientific) supplemented with 10% v/v heat inactivated fetal bovine serum (Life Technologies; ThermoFisher Scientific). Retroviral SARS-CoV-2 pseudovirus were generated by co-transfecting expression plasmids containing SARS-CoV-2 Spike which were kindly provided by Dr Markus Hoffmann, 28 and the MLV gag/pol and luciferase vectors which were kindly provided by Prof. Francois-Loic Cosset, 29,30 in CD81KO 293T cells, which were kindly provided by Dr Joe Grove, 31 using mammalian Calphos transfection kit (Takara Bio). Culture supernatants containing pseudovirus were harvested 48 hours post transfection and clarified of cellular debris by centrifugation at 500xg for 10 minutes.…”

Section: Sars-cov-2 Pseudovirus Neutralisation Assaymentioning

confidence: 99%

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection

Abayasingam

Balachandran

Agapiou³

et al. 2021

Cell Reports Medicine

Self Cite

View full text Add to dashboard Cite

Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 exist, with evidence of antibody titres declining rapidly after infection and reports of reinfection. Here we monitor the antibody responses against SARS-CoV-2 receptor binding domain (RBD) for up to six months after infection. While antibody titres are maintained, about 13% of the cohort’s neutralising responses return to background. However, encouragingly in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire.

show abstract

Section: Sars-cov-2 Pseudovirus Neutralisation Assaymentioning

confidence: 99%

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection

Abayasingam

Balachandran

Agapiou³

et al. 2021

Cell Reports Medicine

Self Cite

View full text Add to dashboard Cite

show abstract

“…CD63 and CD81 are members of the tetraspanin family of proteins, naturally expressed on HEK293 cells and on most other cell types, at modest levels. 32,33 We confirmed by flow cytometry that these monoclonal antibodies bound to HEK293 cells (Fig. S3, ESI †).…”

Section: Intercellular Receptor Activation Assaysmentioning

confidence: 61%

Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

et al. 2021

View full text Add to dashboard Cite

show abstract

“…Pseudovirus neutralisation assays were performed as previously described. 36 In brief, SARS-CoV-2 pseudo-particles were generated by co-transfecting expression plasmids containing SARS-CoV-2 Spike (kindly provided by Dr Markus Hoffmann) 37 and the MLV gag/pol and luciferase vectors (kindly provided by Prof. Francois-Loic Cosset) 38,39 in CD81KO 293T cells (kindly provided by Dr Joe Grove), 40 using mammalian Calphos transfection kit (Takara Bio). Transfected cells were then incubated at 32°C, 5% CO 2 with the culture supernatants containing SARS-CoV-2 virus pseudoparticles (SARS-2pp) and harvested 48 hours post-transfection, concentrated 10-fold using 100,000 MWCO Vivaspin centrifugal concentrators (Sartorius) and stored at –80°C.…”

Section: Methodsmentioning

confidence: 99%

Section: Sars-cov-2 Pseudovirus Neutralisation Assaymentioning

confidence: 99%

COVID-19 convalescents exhibit deficient humoral and T cell responses to variant of concern Spike antigens at 12 month post-infection

García-Valtanen

Hope

Masavuli

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

BackgroundThe duration and magnitude of SARS-CoV-2 immunity after infection, especially with regard to the emergence of new variants of concern (VoC), remains unclear. Here, immune memory to primary infection and immunity to VoC was assessed in mild-COVID-19 convalescents one year after infection and in the absence of viral re-exposure or COVID-19 vaccination.MethodsSerum and PBMC were collected from mild-COVID-19 convalescents at ∼6 and 12 months after a COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15-40). Serum titers of RBD and Spike-specific Ig were quantified by ELISA. Virus neutralisation was assessed against homologous, pseudotyped virus and homologous and VoC live viruses. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped by activation-induced marker assay, while T cell functionality was assessed by intracellular cytokine staining using peptides specific to homologous Spike virus antigen and four VoC Spike antigens.FindingsAt 12 months after mild-COVID-19, >90% of convalescents remained seropositive for RBD-IgG and 88.9% had circulating RBD-specific memory B cells. Despite this, only 51.2% convalescents had serum neutralising activity against homologous live-SARS-CoV-2 virus, which decreased to 44.2% when tested against live B.1.1.7, 4.6% against B.1.351, 11.6% against P.1 and 16.2%, against B.1.617.2 VoC. Spike and non-Spike-specific T cells were detected in >50% of convalescents with frequency values higher for Spike antigen (95% CI, 0.29-0.68% in CD4+ and 0.11-0.35% in CD8+ T cells), compared to non-Spike antigens. Despite the high prevalence and maintenance of Spike-specific T cells in Spike ‘high-responder’ convalescents at 12 months, T cell functionality, measured by cytokine expression after stimulation with Spike epitopes corresponding to VoC was severely affected.InterpretationsSARS-CoV-2 immunity is retained in a significant proportion of mild COVID-19 convalescents 12 months post-infection in the absence of re-exposure to the virus. Despite this, changes in the amino acid sequence of the Spike antigen that are present in current VoC result in virus evasion of neutralising antibodies, as well as evasion of functional T cell responses.FundingThis work was funded by project grants from The Hospital Research Foundation and Women’s and Children’s Hospital Foundation, Adelaide, Australia. MGM is THRF Early Career Fellow. BGB is THRF Mid-Career Fellow. This project has been supported partly with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016 to A.S. and Contract No. 75N9301900065 to A.S, D.W.Evidence before this studyWe regularly searched on PubMed and Google Scholar in June-October 2021 using individual or combinations of the terms “long-term immunity”, “SARS-CoV-2”, “antigenic breadth”, “variant of concern” and “COVID-19”. We found studies that had assessed immune correlates at multipe time points after COVID-19 disease onset in convalescents, but not the antigenic breadth of T cells and antibodies and not in relation to VoC. Other immune studies in virus naive vaccinees, or vaccinated convalescents evaluated VoC-specific immunity, but not in convalescents that have not been vaccinated. In summary, we could not find long-term studies providing and in-depth evaluation of functionality of humoral and cell-mediated immunity, combined with addressing the adaptability of these immune players to VoC.Added value of this studyThe window of opportunity to conduct studies in COVID-19 convalescents (i.e. natural immunity to SARS-CoV-2) is closing due to mass vaccination programs. Here, in a cohort of unvaccinated mild-COVID-19 convalescents, we conducted a comprehensive, longitudinal, long-term immune study, which included functional assays to assess immune fitness against antigenically different VoC. Importantly, the cohort resided in a SARS-CoV-2-free community for the duration of the study with no subsequent re-exposure or infection. Our findings reveal a deeply weakened humoral response and functional vulnerability of T cell responses to VoC Spike antigens.Implications of all the available evidenceThis study provides a valuable snapshot of the quality of SARS-CoV-2 natural immunity and its durability in the context of a pandemic in which new variants continuously emerge and challenge pre-existing immune responses in convalescents and vacinees. Our results serve as a warning that delays in vaccination programs could lead to an increase in re-infection rates of COVID-19 convalescents, caused by virus variants that escape humoral and cell-mediated immune responses. Furthermore, they reinforce the potential benefit of booster vaccination that is tuned to the active variants.

show abstract

Optimised cell systems for the investigation of hepatitis C virus E1E2 glycoproteins

Cited by 6 publications

References 72 publications

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection

Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

COVID-19 convalescents exhibit deficient humoral and T cell responses to variant of concern Spike antigens at 12 month post-infection

Contact Info

Product

Resources

About