Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

The P681H Mutation in the Spike Glycoprotein of the Alpha Variant of SARS-CoV-2 Escapes IFITM Restriction and Is Necessary for Type I Interferon Resistance.

The appearance of new dominant variants of concern (VOC) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) threatens the global response to the coronavirus disease 2019 (COVID-19) pandemic. Of these, the alpha variant (also known as B.1.1.7), which appeared initially in the United Kingdom, became the dominant variant in much of Europe and North America in the first half of 2021. The spike (S) glycoprotein of alpha acquired seven mutations and two deletions compared to the ancestral virus, including the P681H mutation adjacent to the polybasic cleavage site, which has been suggested to enhance S cleavage. Here, we show that the alpha spike protein confers a level of resistance to beta interferon (IFN-ß) in human lung epithelial cells. This correlates with resistance to an entry restriction mediated by interferon-induced transmembrane protein 2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is essential for resistance to IFN-ß and context-dependent resistance to IFITMs in the alpha S. P681H reduces dependence on endosomal cathepsins, consistent with enhanced cell surface entry. However, reversion of H681 does not reduce cleaved spike incorporation into particles, indicating that it exerts its effect on entry and IFN-ß downstream of furin cleavage. Overall, we suggest that, in addition to adaptive immune escape, mutations associated with VOC may well also confer a replication and/or transmission advantage through adaptation to resist innate immune mechanisms. IMPORTANCE Accumulating evidence suggests that variants of concern (VOC) of SARS-CoV-2 evolve to evade the human immune response, with much interest focused on mutations in the spike protein that escape from antibodies. However, resistance to the innate immune response is essential for efficient viral replication and transmission. Here, we show that the alpha (B.1.1.7) VOC of SARS-CoV-2 is substantially more resistant to type I interferons than the parental Wuhan-like virus. This correlates with resistance to the antiviral protein IFITM2 and enhancement by its paralogue IFITM3. The key determinant of this is a proline-to-histidine change at position 681 in S adjacent to the furin cleavage site, which in the context of the alpha spike modulates cell entry pathways of SARS-CoV-2. Reversion of the mutation is sufficient to restore interferon and IFITM2 sensitivity, highlighting the dynamic nature of the SARS CoV-2 as it adapts to both innate and adaptive immunity in the humans.

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction.

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.

Combined epidemiological and genomic analysis of nosocomial SARS-CoV-2 infection early in the pandemic and the role of unidentified cases in transmission.

OBJECTIVES: To analyse nosocomial transmission in the early stages of the coronavirus 2019 (COVID-19) pandemic at a large multisite healthcare institution. Nosocomial incidence is linked with infection control interventions. METHODS: Viral genome sequence and epidemiological data were analysed for 574 consecutive patients, including 86 nosocomial cases, with a positive PCR test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first 19 days of the pandemic. RESULTS: Forty-four putative transmission clusters were found through epidemiological analysis; these included 234 cases and all 86 nosocomial cases. SARS-CoV-2 genome sequences were obtained from 168/234 (72%) of these cases in epidemiological clusters, including 77/86 nosocomial cases (90%). Only 75/168 (45%) of epidemiologically linked, sequenced cases were not refuted by applying genomic data, creating 14 final clusters accounting for 59/77 sequenced nosocomial cases (77%). Viral haplotypes from these clusters were enriched 1-14x (median 4x) compared to the community. Three factors implicated unidentified cases in transmission: (a) community-onset or indeterminate cases were absent in 7/14 clusters (50%), (b) four clusters (29%) had additional evidence of cryptic transmission, and (c) in three clusters (21%) diagnosis of the earliest case was delayed, which may have facilitated transmission. Nosocomial cases decreased to low levels (0-2 per day) despite continuing high numbers of admissions of community-onset SARS-CoV-2 cases (40-50 per day) and before the impact of introducing universal face masks and banning hospital visitors. CONCLUSION: Genomics was necessary to accurately resolve transmission clusters. Our data support unidentified cases-such as healthcare workers or asymptomatic patients-as important vectors of transmission. Evidence is needed to ascertain whether routine screening increases case ascertainment and limits nosocomial transmission.

Neutralizing antibody activity in convalescent sera from infection in humans with SARS-CoV-2 and variants of concern.

COVID-19 vaccine design and vaccination rollout need to take into account a detailed understanding of antibody durability and cross-neutralizing potential against SARS-CoV-2 and emerging variants of concern (VOCs). Analyses of convalescent sera provide unique insights into antibody longevity and cross-neutralizing activity induced by variant spike proteins, which are putative vaccine candidates. Using sera from 38 individuals infected in wave 1, we show that cross-neutralizing activity can be detected up to 305 days pos onset of symptoms, although sera were less potent against B.1.1.7 (Alpha) and B1.351 (Beta). Over time, despite a reduction in overall neutralization activity, differences in sera neutralization potency against SARS-CoV-2 and the Alpha and Beta variants decreased, which suggests that continued antibody maturation improves tolerance to spike mutations. We also compared the cross-neutralizing activity of wave 1 sera with sera from individuals infected with the Alpha, the Beta or the B.1.617.2 (Delta) variants up to 79 days post onset of symptoms. While these sera neutralize the infecting VOC and parental virus to similar levels, cross-neutralization of different SARS-CoV-2 VOC lineages is reduced. These findings will inform the optimization of vaccines to protect against SARS-CoV-2 variants.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Comparative performance of SARS-CoV-2 lateral flow antigen tests and association with detection of infectious virus in clinical specimens: a single-centre laboratory evaluation study.

BACKGROUND: Lateral flow devices (LFDs) for rapid antigen testing are set to become a cornerstone of SARS-CoV-2 mass community testing, although their reduced sensitivity compared with PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is, therefore, essential for successful implementation. We evaluated six commercial LFDs and assessed their correlation with infectious virus culture and PCR cycle threshold (Ct) values. METHODS: In a single-centre, laboratory evaluation study, we did a head-to-head comparison of six LFDs commercially available in the UK: Innova Rapid SARS-CoV-2 Antigen Test, Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette, E25Bio Rapid Diagnostic Test, Encode SARS-CoV-2 Antigen Rapid Test Device, SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test. We estimated the specificities and sensitivities of the LFDs using stored naso-oropharyngeal swabs collected at St Thomas' Hospital (London, UK) for routine diagnostic SARS-CoV-2 testing by real-time RT-PCR (RT-rtPCR). Swabs were from inpatients and outpatients from all departments of St Thomas' Hospital, and from health-care staff (all departments) and their household contacts. SARS-CoV-2-negative swabs from the same population (confirmed by RT-rtPCR) were used for comparative specificity determinations. All samples were collected between March 23 and Oct 27, 2020. We determined the limit of detection (LOD) for each test using viral plaque-forming units (PFUs) and viral RNA copy numbers of laboratory-grown SARS-CoV-2. Additionally, LFDs were selected to assess the correlation of antigen test result with RT-rtPCR Ct values and positive viral culture in Vero E6 cells. This analysis included longitudinal swabs from five infected inpatients with varying disease severities. Furthermore, the sensitivities of available LFDs were assessed in swabs (n=23; collected from Dec 4, 2020, to Jan 12, 2021) confirmed to be positive (RT-rtPCR and whole-genome sequencing) for the B.1.1.7 variant, which was the dominant genotype in the UK at the time of study completion. FINDINGS: All LFDs showed high specificity (≥98·0%), except for the E25Bio test (86·0% [95% CI 77·9-99·9]), and most tests reliably detected 50 PFU/test (equivalent SARS-CoV-2 N gene Ct value of 23·7, or RNA copy number of 3 × 106/mL). Sensitivities of the LFDs on clinical samples ranged from 65·0% (55·2-73·6) to 89·0% (81·4-93·8). These sensitivities increased to greater than 90% for samples with Ct values of lower than 25 for all tests except the SureScreen fluorescence (SureScreen-F) test. Positive virus culture was identified in 57 (40·4%) of 141 samples; 54 (94·7%) of the positive cultures were from swabs with Ct values lower than 25. Among the three LFDs selected for detailed comparisons (the tests with highest sensitivity [Innova], highest specificity [Encode], and alternative technology [SureScreen-F]), sensitivity of the LFDs increased to at least 94·7% when only including samples with detected viral growth. Longitudinal studies of RT-rtPCR-positive samples (tested with Innova, Encode, and both SureScreen-F and the SureScreen visual [SureScreen-V] test) showed that most of the tests identified all infectious samples as positive. Test performance (assessed for Innova and SureScreen-V) was not affected when reassessed on swabs positive for the UK variant B.1.1.7. INTERPRETATION: In this comprehensive comparison of antigen LFDs and virus infectivity, we found a clear relationship between Ct values, quantitative culture of infectious virus, and antigen LFD positivity in clinical samples. Our data support regular testing of target groups with LFDs to supplement the current PCR testing capacity, which would help to rapidly identify infected individuals in situations in which they would otherwise go undetected. FUNDING: King's Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation, UK Department of Health, National Institute for Health Research Comprehensive Biomedical Research Centre.

Antibody longevity and cross-neutralizing activity following SARS-CoV-2 wave 1 and B.1.1.7 infections.

As SARS-CoV-2 variants continue to emerge globally, a major challenge for COVID-19 vaccination is the generation of a durable antibody response with cross-neutralizing activity against both current and newly emerging viral variants. Cross-neutralizing activity against major variants of concern (B.1.1.7, P.1 and B.1.351) has been observed following vaccination, albeit at a reduced potency, but whether vaccines based on the Spike glycoprotein of these viral variants will produce a superior cross-neutralizing antibody response has not been fully investigated. Here, we used sera from individuals infected in wave 1 in the UK to study the long-term cross-neutralization up to 10 months post onset of symptoms (POS), as well as sera from individuals infected with the B.1.1.7 variant to compare cross-neutralizing activity profiles. We show that neutralizing antibodies with cross-neutralizing activity can be detected from wave 1 up to 10 months POS. Although neutralization of B.1.1.7 and B.1.351 is lower, the difference in neutralization potency decreases at later timepoints suggesting continued antibody maturation and improved tolerance to Spike mutations. Interestingly, we found that B.1.1.7 infection also generates a cross-neutralizing antibody response, which, although still less potent against B.1.351, can neutralize parental wave 1 virus to a similar degree as B.1.1.7. These findings have implications for the optimization of vaccines that protect against newly emerging viral variants.

More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.

Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2.

The cellular entry of severe acute respiratory syndrome-associated coronaviruses types 1 and 2 (SARS-CoV-1 and -2) requires sequential protease processing of the viral spike glycoprotein. The presence of a polybasic cleavage site in SARS-CoV-2 spike at the S1/S2 boundary has been suggested to be a factor in the increased transmissibility of SARS-CoV-2 compared to SARS-CoV-1 by facilitating maturation of the spike precursor by furin-like proteases in the producer cells rather than endosomal cathepsins in the target. We investigate the relevance of the polybasic cleavage site in the route of entry of SARS-CoV-2 and the consequences this has for sensitivity to interferons (IFNs) and, more specifically, the IFN-induced transmembrane (IFITM) protein family that inhibit entry of diverse enveloped viruses. We found that SARS-CoV-2 is restricted predominantly by IFITM2, rather than IFITM3, and the degree of this restriction is governed by route of viral entry. Importantly, removal of the cleavage site in the spike protein renders SARS-CoV-2 entry highly pH and cathepsin dependent in late endosomes, where, like SARS-CoV-1 spike, it is more sensitive to IFITM2 restriction. Furthermore, we found that potent inhibition of SARS-CoV-2 replication by type I but not type II IFNs is alleviated by targeted depletion of IFITM2 expression. We propose that the polybasic cleavage site allows SARS-CoV-2 to mediate viral entry in a pH-independent manner, in part to mitigate against IFITM-mediated restriction and promote replication and transmission. This suggests that therapeutic strategies that target furin-mediated cleavage of SARS-CoV-2 spike may reduce viral replication through the activity of type I IFNs.IMPORTANCE The furin cleavage site in the spike protein is a distinguishing feature of SARS-CoV-2 and has been proposed to be a determinant for the higher transmissibility between individuals, compared to SARS-CoV-1. One explanation for this is that it permits more efficient activation of fusion at or near the cell surface rather than requiring processing in the endosome of the target cell. Here, we show that SARS-CoV-2 is inhibited by antiviral membrane protein IFITM2 and that the sensitivity is exacerbated by deletion of the furin cleavage site, which restricts viral entry to low pH compartments. Furthermore, we find that IFITM2 is a significant effector of the antiviral activity of type I interferons against SARS-CoV-2 replication. We suggest that one role of the furin cleavage site is to reduce SARS-CoV-2 sensitivity to innate immune restriction, and thus, it may represent a potential therapeutic target for COVID-19 treatment development.

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike.

The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans.

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.

Comparative assessment of multiple COVID-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings.

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays-a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)-on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.

Real-world evaluation of a novel technology for quantitative simultaneous antibody detection against multiple SARS-CoV-2 antigens in a cohort of patients presenting with COVID-19 syndrome.

An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(-). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11-48) of initially RNA(-) patients, in 36% (95% CI 17-54) of RNA(+) patients before 10 days, 77% (95% CI 67-87) between 10 and 20 days and 95% (95% CI 86-100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75-95) and 75% specificity (95% CI 22-99), although specificity compared with historical controls was 100% (95%CI 91-100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19.

Retroviral retention activates a Syk-dependent HemITAM in human tetherin.

Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosine kinase (Syk), which is required for downstream NF-κB activation, indicating that the tetherin cytoplasmic tail resembles the hemi-immunoreceptor tyrosine-based activation motifs (hemITAMs) found in C-type lectin pattern recognition receptors. Retroviral-induced tetherin signaling is coupled to the cortical actin cytoskeleton via the Rac-GAP-containing protein RICH2 (ARHGAP44), and a naturally occurring tetherin polymorphism with reduced RICH2 binding exhibits decreased phosphorylation and NF-κB activation. Thus, upon virion retention, this linkage to the actin cytoskeleton likely triggers tetherin phosphorylation and subsequent signal transduction to induce an antiviral state.

Ig-like transcript 7, but not bone marrow stromal cell antigen 2 (also known as HM1.24, tetherin, or CD317), modulates plasmacytoid dendritic cell function in primary human blood leukocytes.

The Ig-like transcript (ILT) 7 is a surface molecule selectively expressed by human plasmacytoid dendritic cells (pDCs). ILT7 cross-linking suppresses pDC activation and type I IFN (IFN-I) secretion following TLR7/9 engagement. The bone marrow stromal cell Ag 2 (BST2, aka HM1.24, tetherin, or CD317) is expressed by different cell types upon exposure to IFN-I and is a natural ligand for ILT7. In this study, we show that ILT7 expression decreased spontaneously in pDCs upon in vitro culture, which correlates with pDC differentiation measured as increased side scatter properties and CCR7 expression. TLR7/9 ligands, as well as HIV, induced BST2 upregulation on all tested cell types except T cells, which required TCR stimulation to respond to TLR9L-induced IFN-I. IFN-γ, IL-4, IL-10, and TNF-α had only marginal effects on BST2 expression in blood leukocytes compared with TLR9L. Preincubation with ILT7 cross-linking Ab inhibited IFN-I production in PBMCs treated with TLR7/9L or HIV, whereas BST2 blockade did not affect IFN-I responses even when BST2 upregulation was further boosted with TCR agonists or immunoregulatory cytokines. Our data indicate that BST2-mediated ILT7 cross-linking may act as a homeostatic regulatory mechanism on immature circulating pDC, rather than a negative feedback for activated mature pDCs that have downregulated ILT7.

Evidence for IFNα-induced, SAMHD1-independent inhibitors of early HIV-1 infection.

BACKGROUND: Type I interferon (IFN) treatment of some cells, including dendritic cells, macrophages and monocytic THP-1 cells, restricts HIV-1 infection and prevents viral cDNA accumulation. Sterile alpha motif and HD domain protein 1 (SAMHD1), a dGTP-regulated deoxynucleotide triphosphohydrolase, reduces HIV-1 infectivity in myeloid cells, likely by limiting dNTPs available for reverse transcription, and has been described as IFNα-inducible. Myeloid cell infection by HIV-1 is enhanced by HIV-2/SIVSM Vpx, which promotes SAMHD1 degradation, or by exogenous deoxyribonucleoside (dN) addition. FINDINGS: SAMHD1 expression was not substantially influenced by IFNα treatment of monocyte-derived macrophages or THP-1 cells. The contributions of SAMHD1 to the inhibition of HIV-1 infectivity by IFNα were assessed through the provision of Vpx, exogenous dN addition, or via RNAi-mediated SAMHD1 knock-down. Both Vpx and dN efficiently restored infection in IFNα-treated macrophages, albeit not to the levels seen with these treatments in the absence of IFNα. Similarly using differentiated THP-1 cells, the addition of Vpx or dNs, or SAMHD1 knock-down, also stimulated infection, but failing to match the levels observed without IFNα. Neither Vpx addition nor SAMHD1 knock-down reversed the IFNα-induced blocks to HIV-1 infection seen in dividing U87-MG or THP-1 cells. Therefore, altered SAMHD1 expression or function cannot account for the IFNα-induced restriction to HIV-1 infection seen in many cells and cell lines. CONCLUSION: IFNα establishes an anti-HIV-1 phenotype in many cell types, and appears to accomplish this without potentiating SAMHD1 function. We conclude that additional IFNα-induced suppressors of the early stages of HIV-1 infection await identification.

Innate sensing of HIV-1 assembly by Tetherin induces NFκB-dependent proinflammatory responses.

Antiviral proteins that recognize pathogen-specific or aberrantly located molecular motifs are perfectly positioned to act as pattern-recognition receptors and signal to the immune system. Here we investigated whether the interferon-induced viral restriction factor tetherin (CD317/BST2), which is known to inhibit HIV-1 particle release by physically tethering virions to the cell surface, has such a signaling role. We find that upon restriction of Vpu-defective HIV-1, tetherin acts as a virus sensor to induce NFκB-dependent proinflammatory gene expression. Signaling requires both tetherin's extracellular domain involved in virion retention and determinants in the cytoplasmic tail, including an endocytic motif, although signaling is independent of virion endocytosis. Furthermore, recruitment of the TNF-receptor-associated factor TRAF6 and activation of the mitogen-activated protein kinase TAK1 are critical for signaling. Human tetherin's ability to mediate efficient signaling may have arisen as a result of a five amino acid deletion that occurred in hominids after their divergence from chimpanzees.