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Coronaviruses infections, culminating in the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic beginning in 2019, have highlighted the importance of effective vaccines to induce an antibody response with cross-neutralizing activity. COVID-19 vaccines have been rapidly developed to reduce the burden of SARS-CoV-2 infections and disease severity. Cross-protection from seasonal human coronaviruses (hCoVs) infections has been hypothesized but is still controversial. Here, we investigated the neutralizing activity against ancestral SARS-CoV-2 and the variants of concern (VOCs) in individuals vaccinated with two doses of either BNT162b2, mRNA-1273, or AZD1222, with or without a history of SARS-CoV-2 infection. Antibody neutralizing activity to SARS-CoV-2 and the VOCs was higher in BNT162b2-vaccinated subjects who were previously infected with SARS-CoV-2 and conferred broad-spectrum protection. The Omicron BA.1 variant was the most resistant among the VOCs. COVID-19 vaccination did not confer protection against hCoV-HKU1. Conversely, antibodies induced by mRNA-1273 vaccination displayed a boosting in their neutralizing activity against hCoV-NL63, whereas AZD1222 vaccination increased antibody neutralization against hCoV-229E, suggesting potential differences in antigenicity and immunogenicity of the different spike constructs used between various vaccination platforms. These data would suggest that there may be shared epitopes between the HCoVs and SARS-CoV-2 spike proteins.

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 (cCPE), 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.

The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune driven antigenic variation and ongoing adaptation to a new host.

The chloride intracellular channel (CLIC) protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains under investigation. Here, cellular Cl− efflux assays and immunofluorescence microscopy studies have identified intracellular Zn2+ release as the trigger for CLIC1 activation and membrane insertion. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing Cl− efflux in a pH-dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH-mediated activation of Cl− efflux.

Funder: UK Research and Innovation; Id: http://dx.doi.org/10.13039/100014013, Funder: National Institute for Health Research; Id: http://dx.doi.org/10.13039/501100000272, The virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the global coronavirus disease-2019 (COVID-19) pandemic, spread rapidly around the world causing high morbidity and mortality. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs), and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2. We use functional, neutralizing assays to investigate cross-reactive antibodies and their relationship with COVID-19 severity. We analyzed the neutralization of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63. We found that although HCoV neutralization was very common there was little evidence that these antibodies neutralized SARS-CoV-2. Despite no evidence in cross-neutralization, levels of NL63 neutralizing antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

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.

International audience; The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

BackgroundThe 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.MethodsUsing 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.FindingsOur 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.InterpretationExpressing our data in IU/ml, we provide a benchmark pre-vaccine standardised dataset that compares disease severity with neutralising antibody titres. Our data may now be compared across multiple laboratories. The continued use and aggregation of standardised data will eventually assist in defining correlates of protection.FundingUKRI and NIHR; grant number G107217Research in contextEvidence before this studyDuring the first wave outbreak, much focus was placed on the role of neutralising antibodies and titres generated upon infection to ancestral SARS-CoV-2. Due to the large amounts of different assays used to elucidate the antibody-mediated immunity and laboratory to laboratory, large amounts of invaluable data could not be directly compared in order to define a correlate of protection, due to variability in the results. The WHO International Standard for anti-SARS-CoV-2 Immunoglobulin sera was made in order to standardise future data so that comparisons may take place.Added value of this studyOur study compares the neutralisation capacity of sera from patients and healthcare workers (HCWs) from the ancestral strain of SARS-CoV-2 against new variants, including the current variants of concern in circulation. We also provide data in International Units per mL, a standardised unitage, for infected individuals that have a clinical severity score, allowing us to assess levels of neutralising antibodies across different severities of COVID-19 disease. By providing a method to calibrate most of the variants of concern so that the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin reagent could be used to standardise our results, therefore making them comparable to other laboratories who also standardised their data in an identical manner.Implications of all the available evidenceContinual use and accumulation of standardised data would eventually lead to defining the correlates of protection against SARS-CoV-2. This could help to inform medical staff to identify which individuals would be a greater risk of a potential reinfection to SARS-CoV-2.

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application. © 2021 Bio-protocol LLC. All Rights Reserved.

This contribution explores in a new statistical perspective the antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 141 coronavirus disease 2019 (COVID-19) patients exhibiting a broad range of clinical manifestations. This cohort accurately reflects the characteristics of the first wave of the SARS-CoV-2 pandemic in Italy. We determined the IgM, IgA, and IgG levels towards SARS-CoV-2 S1, S2, and NP antigens, evaluating their neutralizing activity and relationship with clinical signatures. Moreover, we longitudinally followed 72 patients up to 9 months postsymptoms onset to study the persistence of the levels of antibodies. Our results showed that the majority of COVID-19 patients developed an early virus-specific antibody response. The magnitude and the neutralizing properties of the response were heterogeneous regardless of the severity of the disease. Antibody levels dropped over time, even though spike reactive IgG and IgA were still detectable up to 9 months. Early baseline antibody levels were key drivers of the subsequent antibody production and the long-lasting protection against SARS-CoV-2. Importantly, we identified anti-S1 IgA as a good surrogate marker to predict the clinical course of COVID-19. Characterizing the antibody response after SARS-CoV-2 infection is relevant for the early clinical management of patients as soon as they are diagnosed and for implementing the current vaccination strategies.

The CLIC protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and the plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains unclear. Here, cellular chloride efflux assays and immunofluorescence microscopy studies have identified Zn2+ intracellular release as the trigger for CLIC1 activation and membrane relocalisation. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing chloride efflux in a pH dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH activation of chloride efflux.

The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs) and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2.We use functional, neutralising assays to investigate cross reactive antibodies and their relationship with COVID-19 severity. We analysed neutralisation of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63.We found that although HCoV neutralisation was very common there was little evidence that these antibodies neutralised SARS-CoV-2. Despite no evidence in cross neutralisation, levels of NL63 neutralisating antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely. Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates. Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool. Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed. Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead. Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large 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 as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K 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 immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

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.

Background Neutralizing antibodies are important for protection against the pandemic SARS-CoV-2 virus, and long-term memory responses determine the risk of re-infection or boosting after vaccination. T-cellular responses are considered important for partial protection against novel variants of concern. Methods A prospective cohort of hospitalized (n = 14) and community (n = 38) patients with rt-PCR confirmed SARS-CoV-2 infection were recruited. Blood samples and clinical data were collected when diagnosed and at 6 months. Serum samples were analyzed for SARS-CoV-2-spike specific antibodies using ELISA (IgG, IgA, IgM), pseudotype neutralization and microneutralization assays. Peripheral blood mononuclear cells were investigated for virus-specific T-cell responses in the interferon-γ and interleukin-2 fluorescent-linked immunosorbent spot (FluroSpot) assay. Results We found durable SARS-CoV-2 spike- and internal protein specific T-cellular responses in patients with persistent antibodies at 6 months. Significantly higher IL-2 and IFN-γ secreting T-cell responses as well as SARS-CoV-2 specific IgG and neutralizing antibodies were detected in hospitalized compared to community patients. The immune response was impacted by age, gender, comorbidity and severity of illness, reflecting clinical observations. Conclusions SARS-CoV-2 specific T-cellular and antibody responses persisted for 6 months post confirmed infection. In previously infected patients, re-exposure or vaccination will boost long-term immunity, possibly providing protection against re-infection with variant viruses.

Understanding the levels of immunity in both the immunocompromised and general populations will facilitate the development of public health strategies to contain and prevent novel variants of interest or the emergence of vaccine-resistant variants that could threaten COVID-19 immunisation programmes.

Background A notable feature of coronavirus disease 2019 (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-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. Methods We investigated the presence of cross-reactive anti–SARS-CoV-2 IgG antibodies to spike 1 (S1), S1-receptor-binding domain (S1-RBD), and nucleocapsid protein (NP) by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralization assay, in prepandemic 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 anti–S1-RBD antibody levels correlated with anti–HCoV-HKU1 and anti–HCoV-OC43 S1 antibody titers in prepandemic samples (P < .001). There were marked increases of anti–HCoV-HKU1 and - OC43 S1 (but not anti-NL63 and -229E S1-RBD) antibody titers in serum samples from convalescent COVID-19 patients (P < .001), indicating an activation of cross-reactive immunological memory to β-coronavirus spike. Conclusions We demonstrated cross-reactive anti–SARS-CoV-2 antibodies in prepandemic 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.

A large portion of the UK general population has currently only received a single dose of a COVID-19 vaccination (either BNT162b2 (Pfizer) or AZD1222 (Vaxevria, AstraZeneca)), making it critical that we continue to track and understand the levels of protection against SARS-CoV-2 Variants of Concern (VOCs). We undertook a single-centre cross-sectional study in order to understand the variations in vaccine-induced immunity to the SARS-CoV-2 vaccine strain (B.1) and three major VOCs (B.1.1.7, B.1351 and P.1). We studied a cohort of outpatients with immunodeficiencies (IDPs) and health care workers (HCWs) based at the same critical care tertiary NHS Trust, following a single dose of either BNT162b2 or AZD1222 vaccines. Our serological data revealed either undetectable antibodies or low neutralising antibodies (nAbs) in IDPs, with only 5% and 3% showing detectable neutralisation of B.1.1.7 and B.1.351, respectively. Healthy HCWs without a prior SARS-CoV-2 infection demonstrated a wide range of nAb titres post-vaccination. However, their responses are significantly lower than HCWs with previous SARS-CoV-2 infection. Neutralisation of VOCs with the E484K mutation (B.1.351 and P.1) were consistently lower in HCWs in the absence of evidence of prior SARS-CoV-2 infection (p

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

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.

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+and Zn2+trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+and Zn2+binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENTCLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+and Zn2+binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition.

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein’s primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity., Author summary Between 2014 and 2016, West Africa experienced the largest Ebolavirus outbreak in recorded history. The international containment effort spurred extensive research that is enhancing our understanding of ebolavirus replication and pathogenicity. Much has been learned about the main function of each ebolavirus protein since the discovery of the virus in 1976; however, recent ebolavirus research has led to the discovery of many new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenesis and increasing our understanding of how each component of the virus works. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity.

Although the Golgi apparatus has a conserved morphology of flattened stacked cisternae in the vast majority of eukaryotes, the organelle has lost the stacked organization in several eukaryotic lineages raising the question of what range of morphologies is possible for the Golgi. In order to understand this range of organellar diversity, it is necessary to characterise the Golgi in many different lineages. Here we identify the Golgi apparatus in Naegleria, the first description of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two separate species of Naegleria and transcriptomic support to show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker NgCOPB by fluorescence, identifying membranous structures that can be disrupted by Brefeldin A treatment consistent with Golgi localisation. Confocal and immuno-electron microscopy revealed that NgCOPB is localized to membranous structures consistent with tubules. Our data not only identify the Golgi organelle for the first time in this major eukaryotic lineage, but also provide the rare example of a tubular form of the organelle representing an important sampling point for the comparative understanding of Golgi organellar diversity.