Search

Aims: Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection; however, the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity., Methods and Results: Immunofluorescence of post-mortem patient sections was used to assess pathophysiological aspects of COVID-19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor angiotensin-converting enzyme 2 on pericytes when compared to vascular endothelium and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability, including Notch receptor 3, angiopoietin-2, and TEK. Activation of vascular organoids with interleukin-1β did not have an additive effect on vascular permeability. Spike antigen was detected in some patients' lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and von Willebrand factor (VWF) deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin., Conclusion: Together, our data indicate that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment results in pericyte infection, detachment, damage, and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved.)

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of antibodies to SARS-CoV-2 from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the subject group from whom the sera were obtained. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending the disease status of the subject and on the specific antigen targeted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lamerton, Marcial-Juarez, Faustini, Perez-Toledo, Goodall, Jossi, Newby, Chapple, Dietrich, Veenith, Shields, Harper, Henderson, Rayes, Wraith, Watson, Crispin, Drayson, Richter and Cunningham.)

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe., Competing Interests: Declaration of interests J.S.M. is an inventor on the following U.S. patent applications: no. 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”); no. 62/972,886 (“2019-nCoV Vaccine”); no. 63/032,502 (“Engineered Coronavirus Spike (S) Protein and Methods of Use Thereof”)., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Objective: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers., Design: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020., Setting: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK., Participants: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded., Intervention: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked., Main Outcome Measure: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology., Results: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ 2 =21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02)., Conclusions and Relevance: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices., Competing Interests: Competing interests: MTD reports personal fees from Abingdon Health, outside the submitted work. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The importance of salivary SARS-CoV-2 antibodies, following infection and vaccination, has not been fully established. 875 healthcare workers were sampled during the first wave in 2020 and 66 longitudinally in response to Pfizer BioNTech 162b2 vaccination. We measured SARS-CoV-2 total IgGAM and individual IgG, IgA and IgM antibodies. IgGAM seroprevalence was 39.9%; however, only 34.1% of seropositive individuals also had salivary antibodies. Infection generated serum IgG antibodies in 51.4% and IgA antibodies in 34.1% of individuals. In contrast, the salivary antibody responses were dominated by IgA (30.9% and 12% generating IgA and IgG antibodies, respectively). Post 2nd vaccination dose, in serum, 100% of infection naïve individuals had IgG and 82.8% had IgA responses; in saliva, 65.5% exhibited IgG and 55.2% IgA antibodies. Prior infection enhanced the vaccine antibody response in serum but no such difference was observed in saliva. Strong neutralisation responses were seen for serum 6 months post 2nd-vaccination dose (median 87.1%) compared to low neutralisation responses in saliva (median 1%). Intramuscular vaccination induces significant serum antibodies and to a lesser extent, salivary antibodies; however, salivary antibodies are typically non-neutralising. This study provides further evidence for the need of mucosal vaccines to elicit nasopharyngeal/oral protection. Although saliva is an attractive non-invasive sero-surveillance tool, due to distinct differences between systemic and oral antibody responses, it cannot be used as a proxy for serum antibody measurement. • Largest investigation of SARS-CoV-2 serum and salivary antibodies in 875 healthcare workers. • In-depth profiling of combined anti-Spike IgGAM and individual immunoglobulin classes. • Saliva is not equivalent to serum. [ABSTRACT FROM AUTHOR]

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection. We find raised levels of anti-DSG2 autoantibodies in sera from individuals following severe COVID-19. Staining of post-mortem cardiac tissue from individuals that died from COVID-19 with an anti-DSG2 antibody revealed disruption of the intercalated disc between cardiomyocytes that was consistent with separation of the DSG2 protein homodimer. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection. [ABSTRACT FROM AUTHOR]

Background: The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective: To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods: MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe; severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FceR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry Results: The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion: In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme. [ABSTRACT FROM AUTHOR]

Dried blood spot (DBS) samples can be used for the detection of severe acute respiratory syndrome coronavirus 2 spike antibodies. DBS sampling is comparable to matched serum samples with a relative 98.1% sensitivity and 100% specificity. Thus, DBS sampling offers an alternative for population-wide serologic testing in the coronavirus pandemic. [ABSTRACT FROM AUTHOR]