Identification of inflammatory clusters in long-COVID through analysis of plasma biomarker levels.

Mechanisms underlying long COVID remain poorly understood. Patterns of immunological responses in individuals with long COVID may provide insight into clinical phenotypes. Here we aimed to identify these immunological patterns and study the inflammatory processes ongoing in individuals with long COVID. We applied an unsupervised hierarchical clustering approach to analyze plasma levels of 42 biomarkers measured in individuals with long COVID. Logistic regression models were used to explore associations between biomarker clusters, clinical variables, and symptom phenotypes. In 101 individuals, we identified three inflammatory clusters: a limited immune activation cluster, an innate immune activation cluster, and a systemic immune activation cluster. Membership in these inflammatory clusters did not correlate with individual symptoms or symptom phenotypes, but was associated with clinical variables including age, BMI, and vaccination status. Differences in serologic responses between clusters were also observed. Our results indicate that clinical variables of individuals with long COVID are associated with their inflammatory profiles and can provide insight into the ongoing immune responses.

Impact of vaccination and variants of concern on long COVID clinical phenotypes.

BACKGROUND: Defining patterns of symptoms in long COVID is necessary to advance therapies for this heterogeneous condition. Here we aimed to describe clusters of symptoms in individuals with long COVID and explore the impact of the emergence of variants of concern (VOCs) and vaccination on these clusters. METHODS: In a prospective, multi centre cohort study, individuals with symptoms persisting > 4 weeks from acute COVID-19 were divided into two groups based on timing of acute infection; pre-Alpha VOC, denoted wild type (WT) group and post-Alpha VOC (incorporating alpha and delta dominant periods) denoted VOC group. We used multiple correspondence analysis (MCA) and hierarchical clustering in the WT and VOC groups to identify symptom clusters. We then used logistic regression to explore factors associated with individual symptoms. RESULTS: A total of 417 individuals were included in the analysis, 268 in WT and 149 in VOC groups respectively. In both groups MCA identified three similar clusters; a musculoskeletal (MSK) cluster characterised by joint pain and myalgia, a cardiorespiratory cluster and a less symptomatic cluster. Differences in characteristic symptoms were only seen in the cardiorespiratory cluster where a decrease in the frequency of palpitations (10% vs 34% p = 0.008) and an increase in cough (63% vs 17% p < 0.001) in the VOC compared to WT groups was observed. Analysis of the frequency of individual symptoms showed significantly lower frequency of both chest pain (25% vs 39% p = 0.004) and palpitations (12% vs 32% p < 0.001) in the VOC group compared to the WT group. In adjusted analysis being in the VOC group was significantly associated with a lower odds of both chest pain and palpitations, but vaccination was not associated with these symptoms. CONCLUSION: This study suggests changes in long COVID phenotype in individuals infected later in the pandemic, with less palpitations and chest pain reported. Adjusted analyses suggest that these effects are mediated through introduction of variants rather than an effect from vaccination.

Distinct receptor binding domain IgG thresholds predict protective host immunity across SARS-CoV-2 variants and time.

SARS-CoV-2 neutralising antibodies provide protection against COVID-19. Evidence from early vaccine trials suggested binding antibody thresholds could serve as surrogate markers of neutralising capacity, but whether these thresholds predict sufficient neutralising capacity against variants of concern (VOCs), and whether this is impacted by vaccine or infection history remains unclear. Here we analyse individuals recovered from, vaccinated or with hybrid immunity against SARS-CoV-2. An NT50 ≥ 100 IU confers protection in vaccine trials, however, as VOC induce a reduction in NT50, we use NT50 ≥ 1000 IU as a cut off for WT NT50 that would retain neutralisation against VOC. In unvaccinated convalescent participants, a receptor binding domain (RBD) IgG of 456 BAU/mL predicts an NT50 against WT of 1000 IU with an accuracy of 80% (95%CI 73-86%). This threshold maintains accuracy in determining loss of protective immunity against VOC in two vaccinated cohorts. It predicts an NT50 < 100 IU against Beta with an accuracy of 80% (95%CI 67-89%) in 2 vaccine dose recipients. In booster vaccine recipients with a history of COVID-19 (hybrid immunity), accuracy is 87% (95%CI 77-94%) in determining an NT50 of <100 IU against BA.5. This analysis provides a discrete threshold that could be used in future clinical studies.

Development of a novel medium throughput flow-cytometry based micro-neutralisation test for SARS-CoV-2 with applications in clinical vaccine trials and antibody screening.

Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.1.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1:20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto Vero E6 or Vero E6/TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using non-linear regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin. Both Micro-NT and PRNT achieved comparable NT50 values. Further validation showed adequate correlation with PRNT using a panel of secondary standards of clinical convalescent and vaccinated plasma samples. We found the assay to be reproducible through measuring both repeatability and intermediate precision. Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s <1/20 to >1/5000. We could also characterise immune-escape VOC Beta and Omicron BA.5, achieving fold-reductions in neutralising capacity similar to those published. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials.

Long COVID: Clinical characteristics, proposed pathogenesis and potential therapeutic targets.

The emergence of persistent ill-health in the aftermath of SARS-CoV-2 infection has presented significant challenges to patients, healthcare workers and researchers. Termed long COVID, or post-acute sequelae of COVID-19 (PASC), the symptoms of this condition are highly variable and span multiple body systems. The underlying pathophysiology remains poorly understood, with no therapeutic agents proven to be effective. This narrative review describes predominant clinical features and phenotypes of long COVID alongside the data supporting potential pathogenesis of these phenotypes including ongoing immune dysregulation, viral persistence, endotheliopathy, gastrointestinal microbiome disturbance, autoimmunity, and dysautonomia. Finally, we describe current potential therapies under investigation, as well as future potential therapeutic options based on the proposed pathogenesis research.

Performance and validation of an adaptable multiplex assay for detection of serologic response to SARS-CoV-2 infection or vaccination.

Measurement of quantitative antibody responses are increasingly important in evaluating the immune response to infection and vaccination. In this study we describe the validation of a quantitative, multiplex serologic assay utilising an electrochemiluminescence platform, which measures IgG against the receptor binding domain (RBD), spike S1 and S2 subunits and nucleocapsid antigens of SARS-CoV-2. The assay displayed a sensitivity ranging from 73 to 91% and specificity from 90 to 96% in detecting previous infection with SARS-CoV-2 depending on antigenic target and time since infection, and this assay highly correlated with commercially available assays. The within-plate coefficient of variation ranged from 3.8-3.9% and the inter-plate coefficient of variation from 11 to 13% for each antigen.

Individuals with obesity who survive SARS-CoV-2 infection have preserved antigen-specific T cell frequencies.

OBJECTIVE: Obesity is a major risk factor for severe disease in COVID-19, with increased hospitalization, intensive care unit admission, and mortality. This increased impact of COVID-19 in people with obesity (PWO) is likely driven, in part, by the well-described obesity-induced immune dysregulation. Obesity has also been associated with impaired immune memory in many settings, including weakened responses to hepatitis B, tetanus, rabies, and influenza vaccination. Recently, it was reported that PWO who have COVID-19 have reduced IgG antibody titers with defective neutralizing capabilities. However, it remains unknown whether PWO generate durable T cell immunity to SARS-CoV-2. METHODS: This study investigated SARS-CoV-2-specific T cell responses in a cohort of 40 patients (n = 20 PWO and n = 20 matched control individuals) who had recovered from COVID-19. T cell (CD4+ , CD8+ ) cytokine responses (IFNγ, TNFα) to SARS-CoV-2 peptide pools (spike, membrane) were determined using multicolor flow cytometry. RESULTS: Circulating T cells specific for SARS-CoV-2 were readily detected in the total cohort. PWO displayed comparable levels of SARS-CoV-2 spike- and membrane-specific T cells, with both T cell subsets responding. CONCLUSIONS: These data indicate that PWO who survive COVID-19 generate robust and durable SARS-CoV-2-specific T cell immunity that is equivalent to that seen in those without obesity.

Markers of fungal translocation are elevated during post-acute sequelae of SARS-CoV-2 and induce NF-κB signaling.

Long COVID, a type of post-acute sequelae of SARS-CoV-2 (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the mechanisms that drive this inflammation remain unknown. Inflammation during acute coronavirus disease 2019 could be exacerbated by microbial translocation (from the gut and/or lung) to blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We did not observe a significant elevation in plasma markers of bacterial translocation during PASC. However, we observed higher levels of fungal translocation - measured as ß-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared with those without PASC or SARS-CoV-2-negative controls. The higher ß-glucan correlated with higher inflammation and elevated levels of host metabolites involved in activating N-methyl-d-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neurotoxic properties. Mechanistically, ß-glucan can directly induce inflammation by binding to myeloid cells (via Dectin-1) and activating Syk/NF-κB signaling. Using a Dectin-1/NF-κB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-κB signaling compared with plasma from negative controls. This higher NF-κB signaling was abrogated by piceatannol (Syk inhibitor). These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.

Identification of Distinct Long COVID Clinical Phenotypes Through Cluster Analysis of Self-Reported Symptoms.

Background: We aimed to describe the clinical presentation of individuals presenting with prolonged recovery from coronavirus disease 2019 (COVID-19), known as long COVID. Methods: This was an analysis within a multicenter, prospective cohort study of individuals with a confirmed diagnosis of COVID-19 and persistent symptoms >4 weeks from onset of acute symptoms. We performed a multiple correspondence analysis (MCA) on the most common self-reported symptoms and hierarchical clustering on the results of the MCA to identify symptom clusters. Results: Two hundred thirty-three individuals were included in the analysis; the median age of the cohort was 43 (interquartile range [IQR], 36-54) years, 74% were women, and 77.3% reported a mild initial illness. MCA and hierarchical clustering revealed 3 clusters. Cluster 1 had predominantly pain symptoms with a higher proportion of joint pain, myalgia, and headache; cluster 2 had a preponderance of cardiovascular symptoms with prominent chest pain, shortness of breath, and palpitations; and cluster 3 had significantly fewer symptoms than the other clusters (2 [IQR, 2-3] symptoms per individual in cluster 3 vs 6 [IQR, 5-7] and 4 [IQR, 3-5] in clusters 1 and 2, respectively; P < .001). Clusters 1 and 2 had greater functional impairment, demonstrated by significantly longer work absence, higher dyspnea scores, and lower scores in SF-36 domains of general health, physical functioning, and role limitation due to physical functioning and social functioning. Conclusions: Clusters of symptoms are evident in long COVID patients that are associated with functional impairments and may point to distinct underlying pathophysiologic mechanisms of disease.

Immune Dysregulation in Acute SARS-CoV-2 Infection.

Introduction: Neutralizing antibodies have been shown to develop rapidly following SARS-CoV-2 infection, specifically against spike (S) protein, where cytokine release and production is understood to drive the humoral immune response during acute infection. Thus, we evaluated the quantity and function of antibodies across disease severities and analyzed the associated inflammatory and coagulation pathways to identify acute markers that correlate with antibody response following infection. Methods: Blood samples were collected from patients at time of diagnostic SARS-CoV-2 PCR testing between March 2020-November 2020. Plasma samples were analyzed using the MesoScale Discovery (MSD) Platform using the COVID-19 Serology Kit and U-Plex 8 analyte multiplex plate to measure anti-alpha and beta coronavirus antibody concentration and ACE2 blocking function, as well as plasma cytokines. Results: A total of 230 (181 unique patients) samples were analyzed across the 5 COVID-19 disease severities. We found that antibody quantity directly correlated with functional ability to block virus binding to membrane-bound ACE2, where a lower SARS-CoV-2 anti-spike/anti-RBD response corresponded with a lower antibody blocking potential compared to higher antibody response (anti-S1 r = 0.884, P < 0.001; anti-RBD r = 0.75, P < 0.001). Across all the soluble proinflammatory markers we examined, ICAM, IL-1ß, IL-4, IL-6, TNFα, and Syndecan showed a statistically significant positive correlation between cytokine or epithelial marker and antibody quantity regardless of COVID-19 disease severity. Analysis of autoantibodies against type 1 interferon was not shown to be statistically significant between disease severity groups. Conclusion: Previous studies have shown that proinflammatory markers, including IL-6, IL-8, IL-1ß, and TNFα, are significant predictors of COVID-19 disease severity, regardless of demographics or comorbidities. Our study demonstrated that not only are these proinflammatory markers, as well as IL-4, ICAM, and Syndecan, correlative of disease severity, they are also correlative of antibody quantity and quality following SARS-CoV-2 exposure.

Dynamic Change and Clinical Relevance of Postinfectious SARS-CoV-2 Antibody Responses.

BACKGROUND: Although reports suggest that most individuals with coronavirus disease 2019 (COVID-19) develop detectable antibodies postinfection, the kinetics, durability, and relative differences between immunoglobulin M (IgM) and immunoglobulin G (IgG) responses beyond the first few weeks after symptom onset remain poorly understood. METHODS: Within a large, well-phenotyped, diverse, prospective cohort of subjects with and without severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR)-confirmed infection and historical controls derived from cohorts with high prevalence of viral coinfections and samples taken during prior flu seasons, we measured SARS-CoV-2 serological responses (both IgG and IgM) using commercially available assays. We calculated sensitivity, specificity, and relationship with disease severity and mapped the kinetics of antibody responses over time using generalized additive models. RESULTS: We analyzed 1001 samples from 752 subjects, 327 with confirmed SARS-CoV-2 (29.7% with severe disease) spanning a period of 90 days from symptom onset. Sensitivity was lower (44.1%-47.1%) early (<10 days) after symptom onset but increased to >80% after 10 days. IgM positivity increased earlier than IgG-targeted assays, but positivity peaked between days 32 and 38 post-onset of symptoms and declined thereafter, a dynamic that was confirmed when antibody levels were analyzed, with a more rapid decline observed with IgM. Early (<10 days) IgM but not IgG levels were significantly higher in those who subsequently developed severe disease (signal/cutoff 4.20 [0.75-17.93] vs 1.07 [0.21-5.46]; P = .048). CONCLUSIONS: This study suggests that postinfectious antibody responses in those with confirmed COVID-19 begin to decline relatively early postinfection and suggests a potential role for higher IgM levels early in infection in the prediction of subsequent disease severity.

Tocilizumab for the treatment of non-critical COVID-19 pneumonia: an overview of the rationale and clinical evidence to date.

Introduction: Tocilizumab is one of the main repurposed therapies investigated for COVID-19 pneumonia since the start of the pandemic, but there has been conflicting evidence for its use.Areas covered: This review covers the physiology of interleukin-6 and its role in the pathophysiology of COVID-19. We discuss the use of tocilizumab in other diseases and the rationale for its use in COVID-19. We summarize the design, contrasting results, and implications of the clinical trials of tocilizumab in COVID-19 to date and discuss the current guidance for its use.Expert opinion: The evidence to date suggests benefit with the use of tocilizumab in some but not all patients with COVID-19. Benefit seems to be greatest when given early after clinical deterioration with the presence of systemic inflammation. However, questions remain around the optimal timing, patient selection, and concomitant treatments.

COVID19- clinical presentation and therapeutic considerations.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a pandemic infection in 2020 has presented many therapeutic challenges. Not least among these is the importance of abnormal host response to infection that is one of the main drivers of more severe disease. Despite significant research endeavours, very few effective therapies have been identified, in part related to the different pathogenic mechanisms underlying different stages of clinical COVID-19. This mini review summarises data related to current and potential future therapies for COVID-19 and highlights the many challenges inherent in developing effective therapeutic options for new pandemic infection.

A case of Verruconis gallopava infection in a heart transplant recipient successfully treated with posaconazole.

Verruconis gallopava is an environmental dematiaceous fungus that is recognized increasingly as a cause of human disease, especially for immunocompromised persons. Infection can range from superficial and localized lesions to pulmonary involvement and disseminated disease, including central nervous system abscesses. Optimal therapy is undefined. We report a patient post cardiac transplant who had pulmonary infection with V gallopava and was treated successfully with posaconazole.

Successful reconstruction of an ocular defect resulting from granulomatosis with polyangiitis, following treatment with rituximab.

PURPOSE: To report a unique case of orbital inflammatory disease which was ultimately diagnosed as granulomatosis with polyangitis (GPA) and thus successfully treated. OBSERVATION: A 47 year-old man presented with a rapidly progressive necrotic soft tissue mass within the medial antero-superior aspect of the right eyelid and orbit. He also had transient retinal vasculitis in the left. Serology, histology and imaging were atypical of, but consistent with, GPA. He was thus successfully treated with intravenous rituximab followed by reconstruction of the medial eyelid. CONCLUSION AND IMPORTANCE: A high index of suspicion of GPA is required in orbital inflammatory disease, especially when typical diagnostic findings are absent.