34 research outputs found
The Role of Pseudotype Neutralisation Assays in Understanding SARS CoV-2
Neutralisation assays are crucial tools to quantify the presence of functional neutralising antibodies in serum samples. Since the SARS-CoV-2 virus (the causative agent of COVID-19) is designated as a category 3 biosafety level pathogen, pseudotyped viruses bearing the SARS-CoV-2 spike protein permit extensive and widespread serum/plasma screening in a BSL 2 laboratory. These assays can be used to assess viral tropism, vaccine immunogenicity, efficacy of antiviral compounds (incl. therapeutic mAbs) and serosurveillance studies. In this article, we highlight approaches to SARS-CoV-2 viral pseudotyping, its practicality, and utility in increasing our understanding of neutralising antibodies against SARS-CoV-2
Capture and inactivation of viral particles from bioaerosols by electrostatic precipitation.
Infectious viral particles in bioaerosols generated during laparoscopic surgery place staff and patients at significant risk of infection and contributed to the postponement of countless surgical procedures during the COVID-19 pandemic causing excess deaths. The implementation of devices that inactivate viral particles from bioaerosols aid in preventing nosocomial viral spread. We evaluated whether electrostatic precipitation (EP) is effective in capturing and inactivating aerosolised enveloped and non-enveloped viruses. Using a closed-system model mimicking release of bioaerosols during laparoscopic surgery, known concentrations of each virus were aerosolised, exposed to EP and collected for analysis. We demonstrate that both enveloped and non-enveloped viral particles were efficiently captured and inactivated by EP, which was enhanced by increasing the voltage to 10kV or using two discharge electrodes together at 8kV. This study highlights EP as an effective means for capturing and inactivating viral particles in bioaerosols, which may enable continued surgical procedures during future pandemics
Comparison of lentiviral and vesicular stomatitis virus core SARS-CoV-2 pseudotypes and generation of a stable cell line for use in antibody neutralisation assays
Betacoronavirus SARS-CoV-2, the causative agent of COVID19, is a single stranded positive sense RNA virus. Since its emergence there has been great efforts to identify correlates of protection,which is crucial for vaccine evaluation studies. However, handling SARS-CoV-2 requires BSL-3 containment facilities slowing research efforts. Pseudotype viruses (PV) are a safe alternative to authentic virus that can be handled at low containment. PVs are chimeric viruses containing the core of a virus where its genome has been completely or partially replaced by a reporter gene, displaying a correctly folded SARS-CoV-2 spike on its surface. We developed lentiviral and vesicular stomatitis virus (VSV) core PVs alongside a stable A549 cell line expressing receptor ACE2 and protease TMPRSS2 responsible for S protein priming, for use in neutralization assays. Lentiviral PVs were generated by transfection with plasmids encoding the spike, HIV-1 gag-pol and a luciferase reporter. For VSV PVs, producer cells pre-transfected with the spike were infected with recombinant VSV expressing luciferase,before harvesting. The stable A549 cell line was generated by sequential infection of VSV-G PVs bearing lentiviral vectors encoding ACE2 and TMPRSS2 genes followed by antibiotic selection, before being tested in neutralization assays. We compared lentiviral and VSV PV platforms using monoclonal antibodies and convalescent sera with our stable A549 cells or HEK293T cells pre-transfected with plasmids encoding ACE2 and TMPRSS2. Antibody titres showed equivalence however VSV had the advantage of a shorter incubation therefore enabling a higher throughput. PVs offer a robust platform for future seroepidemiology and vaccine evaluation studies
Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.
The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination
Different decay of antibody response and VOC sensitivity in naĂŻve and previously infected subjects at 15 weeks following vaccination with BNT162b2
Background: COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. Methods: Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and the SARS-CoV-2 variants of concern emerged later, respectively. The Wilcoxon matched pair signed rank test and the Kruskal–Wallis test with Dunn’s correction for multiple comparison were applied when needed. Results: Although after 15 weeks from vaccination IgG-RBD dropped in all participants, naïve subjects experienced a more dramatic decline than those with previous SARS-CoV-2 infection. Neutralizing antibodies remained higher in subjects with SARS-CoV-2 history and conferred broad-spectrum protection. Conclusions: These data suggest that hybrid immunity to SARS-CoV-2 has a relevant impact on the development of IgG-RBD upon vaccination. However, the rapid decay of vaccination-elicited antibodies highlights that the administration of a third dose is expected to boost the response and acquire high levels of cross-neutralizing antibodies
Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein
The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1β, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response
Centenarians, semi and supercentenarians, COVID-19 and Spanish flu: a serological assessment to gain insight into the resilience of older centenarians to COVID-19
Background: Although it is well known that the older people have been the most susceptible to COVID-19, there are conflicting data on the susceptibility of centenarians. Two epidemiological study have shown that older centenarians (> 101 years old at the time of the 2020 pandemic peak) are more resilient than the remaining centenarians, suggesting that this resilience might be linked to the 1918 Spanish Flu pandemic. To gain insight into this matter, specifically whether the resilience of older centenarians to SARS-CoV-2 infection is linked to the Spanish Flu they had been affected by, we conducted a retrospective serological study. This study examined serum samples from 33 centenarians, encompassing semi- (aged > 104 109 years, N = 4), born between 1905 and 1922, against both SARS-CoV-2 and 1918 H1N1 pseudotype virus. Results: Anamnestic and laboratory data suggest that SARS-CoV-2 infection occurred in 8 centenarians. The infection appeared to have been asymptomatic or mild, and hospitalization was not required, despite 3 out of 8 being between 109 and 110 years old. The levels of anti-spike antibodies in centenarians infected and/or vaccinated were higher, although not significantly, than those produced by a random sample of seventy-year-old individuals used as controls. All centenarians had antibody levels against the 1918 H1N1 virus significantly higher (almost 50 times) than those observed in the quoted group of seventy-year-old subjects, confirming the key role in maintaining immunological memory from a priming that occurred over 100 years ago. Centenarians whose blood was collected prior to the pandemic outbreak demonstrated neutralising antibodies against the 1918 H1N1 virus, but all these subjects tested negative for SARS-CoV-2. Conclusion: This retrospective study shows that older centenarians are quite resilient to COVID-19, as they are capable of producing good levels of neutralising antibodies and experiencing mild or asymptomatic disease. This could be attributed to the 1918 Spanish flu pandemic through mechanisms other than the presence of cross-reactive antibodies between the 1918 H1N1 virus and SARS-CoV-2. Another possibility is that the association is purely temporal, solely correlated with the advanced age of resilient centenarians compared to those born after 1918, since older centenarians are known to have better control of immune-inflammatory responses
Correlation between pseudotyped virus and authentic virus neutralisation assays, a systematic review and meta-analysis of the literature
Background: The virus neutralization assay is a principal method to assess the efficacy of antibodies in blocking viral entry. Due to biosafety handling requirements of viruses classified as hazard group 3 or 4, pseudotyped viruses can be used as a safer alternative. However, it is often queried how well the results derived from pseudotyped viruses correlate with authentic virus. This systematic review and meta-analysis was designed to comprehensively evaluate the correlation between the two assays.
Methods: Using PubMed and Google Scholar, reports that incorporated neutralisation assays with both pseudotyped virus, authentic virus, and the application of a mathematical formula to assess the relationship between the results, were selected for review. Our searches identified 67 reports, of which 22 underwent a three-level meta-analysis.
Results: The three-level meta-analysis revealed a high level of correlation between pseudotyped viruses and authentic viruses when used in an neutralisation assay. Reports that were not included in the meta-analysis also showed a high degree of correlation, with the exception of lentiviral-based pseudotyped Ebola viruses.
Conclusion: Pseudotyped viruses identified in this report can be used as a surrogate for authentic virus, though care must be taken in considering which pseudotype core to use when generating new uncharacterised pseudotyped viruses
Novel intranasal vaccine targeting SARS-CoV-2 receptor binding domain to mucosal microfold cells and adjuvanted with TLR3 agonist Riboxxim™ elicits strong antibody and T-cell responses in mice
SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin, which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza
Detection of serum cross-reactive antibodies and memory response to SARS-CoV-2 in pre-pandemic and post-COVID-19 convalescent samples
Background
A notable feature of COVID-19 is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-SARS-CoV-2 antibodies.
Methods
We investigated the presence of cross-reactive anti-SARS-CoV-2 IgG antibodies (to spike(S)1, S1-receptor-binding receptor (S1-RBD) and nucleocapsid protein(NP)), by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralisation assay, in pre-pandemic sera collected from children(n=50) and adults(n=45), and compared with serum samples from convalescent COVID-19 patients(n=16).
Results
A significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD and NP antigens, and the anti-S1 and -S1-RBD antibody levels correlated with anti-HCoV-HKU1 and -OC43 S1 antibody titers in pre-pandemic samples(p<0.001). There were marked increases of anti-HCoV-HKU1 and -OC43 S1 (but not anti-NL63 and -229E S-RBD) antibody titres in serum samples from convalescent COVID-19 patients(p<0.001), indicating an activation of cross-reactive immunological memory to β-coronavirus spike.
Conclusions
We demonstrated cross-reactive anti-SARS-CoV-2 antibodies in pre-pandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses