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Background: Severe COVID-19 and obesity are characterized by higher inflammation. We aimed to examine early inflammatory patterns in people with (Ob) and without (NOb) obesity and COVID-19 and how they relate to COVID-19 disease severity Methods: Ob (BMI >30 Kg/m2) and NOb with COVID-19 matched for age, sex and WHO disease severity provided blood early after diagnosis. Immunoassays measured 57 plasma biomarkers reflecting innate immune and endothelial activation, systemic inflammation, coagulation, metabolism and microbial translocation (Fig 1). Between-group differences were assessed by Mann- Whitney. Associations between subsequent maximal COVID-19 severity (mild vs moderate/severe/critical) and biomarkers were explored by logistic regression adjusted for age, sex, hypertension (HTN) and diabetes (DM). Data are median pg/mL [IQR] or n [%] unless stated Results: Of 100 subjects (50 Ob and 50 Nob) presenting between April 2020 and March 2021, characteristics (Ob vs Nob) included: age 65 [23-91] vs 65 [21-95];female sex 27 (48%) vs 28 (56%);BMI 33.7 [30.0-71.8] vs 23.3 [15.3-25.9];disease severity mild 22 [48%] vs 23 [46%], moderate 15 [30%] vs 13 [26%], severe 6 [12%] vs 7 [14%];HTN 30 (60%) vs 17 (34%);DM 19 [38%] vs 6 [12%];days from symptom onset 7 [2-17] vs 8 [1-15];vaccinated 3 (6%) vs 0 (0%). Compared to NOb, Ob had higher IFN-alpha (1.8 [0.6;11] vs 0.9 [0.1;4.7]), CRP (10 mAU/mL [9.6;10.2] vs 9.7 [7.2;10]), IL-1RA (197 [122;399] vs 138 [88;253]), IL-4 (288 AU/mL [161;424] vs 205 [82;333]), vWF (252 [166;383] vs 163 [96;318]), Zonulin (114 ng/mL [77;131] vs 57 [18;106]), Resistin (956 [569;1153] vs 727 [712;1525]), Leptin (3482 [1513;5738] vs 848 [249;2114]), and lower Adiponectin (1.12 mg/L [0.09;1.5] vs 1.5 [1.18;1.93]), all p< 0.05. In both groups higher, proinflammatory IL-18 and lower levels of antiinflammatory CCL22 and IL-5 were associated with higher odds of disease severity, and lower E-selectin with higher disease severity only in Ob. However, in NOb higher type 3 interferons (IL-28A), macrophage activation (sCD163, CCL3) and vascular inflammation markers (ICAM-1, VCAM-1), along with higher S100B, GM-CSF and leptin were also associated with disease severity, a pattern not observed in Ob (Fig 1) Conclusion(s): Although Ob had higher overall levels of inflammation than NOb, few biomarkers predicted subsequent COVID-19 severity in Ob. These differential inflammatory patterns suggest dysregulated immune responses in Ob with COVID-19. (Figure Presented).
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Background: SARS CoV 2 infection alters the immunological profiles of natural killer (NK) cells. However, whether NK anti-viral functions (direct cytotoxicity and/or antibody-dependent cell cytotoxicity (ADCC)) are impaired during severe COVID-19 and what host factors modulate these functions remain unclear. Method(s): Using functional assays, we examined the ability of NK cells from SARS-CoV-2 negative controls (n=12), mild COVID-19 patients (n=26), and hospitalized COVID-19 patients (n=41) to elicit direct cytotoxicity and ADCC [NK degranulation by flow] against cells expressing SARS-CoV-2 antigens. SARS-CoV- 2 N antigen plasma load was measured using an ultra-sensitive Simoa assay. We also phenotypically characterized the baseline expression of NK activating (CD16 and NKG2C), maturation (CD57), and inhibitory (NKG2A and the glyco-immune negative checkpoint Siglec-9) by flow cytometry. Finally, an anti-Siglec-9 blocking antibody was used to examine the impact of Siglec-9 expression on anti-SARS-CoV-2-specific ADCC [degranulation and target cell lysis]. Result(s): NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2-antigen-expressing cells (in direct cytolytic and ADCC assays) than did cells from mild COVID-19 patients or negative controls (Fig. 1A). The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 N-antigen (P<=0.02). Phenotypic and functional analyses showed that NK cells expressing Siglec-9 elicited higher ADCC than Siglec-9- NK cells (P<0.05;Fig. 1B). Consistently, Siglec-9+ NK cells expressed an activated and mature phenotype with higher expression of CD16, CD57, and NKG2C, and lower expression of NKG2A, than Siglec-9- NK cells (P<=0.03). These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells (P<=0.05;Fig. 1C). Conclusion(s): These data support a model (Fig. 1D) in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity can further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. (Figure Presented).
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Background: Neurocognitive dysfunction is common in long COVID and in people living with HIV (PWH). It is unknown whether PWH experience different disturbances in neurocognitive function following COVID-19 compared to HIVseronegative people. Method(s): The amfAR-Johns Hopkins University COVID Recovery Study is a prospective observational cohort study consisting of four groups: participants who had SARS-CoV-2 infection for the first time within 30 days prior to enrollment with HIV (PWH, arm 1) and without HIV (arm 2);participants with no history of SARS-CoV-2 infection with HIV (arm 3) and without HIV (arm 4). 93.5% of the cohort had received a COVID-19 vaccine prior to enrollment. Cognitive tests were administered at 1-and 4-months post symptom onset (arms 1-2) or post-enrollment (arms 3-4) in seven domains. Age standardized scores (all tests) and age-sex-and education-standardized scores (verbal fluency) were obtained. Standardized scores were compared using the Mann-Whitney U Test and the Kruskal-Wallis test. Result(s): PWH scored lower than HIV-seronegative participants at 1 and 4 months post-COVID on three tests: the Hopkins Verbal Learning Test-Revised (HVLT-R) learning (M1, p=0.011, M4, p=0.015), HVLT-R memory (M1, p=0.029, M4, p=0.007), and category-cued verbal fluency (VF;M1&4, p< 0.001). For the majority of timepoints, PWH who were post-COVID produced equivalent scores as PWH who never had COVID (p-levels > 0.05). Comparing post-COVID HIV-seronegative people to those who never had COVID, post-COVID participants scored lower than never-COVID participants on the Oral Trail Making Test part A (OTMT) test of processing speed at month 1 (p=0.033). Between month 1 and 4, HIV-seronegative people who were post-COVID showed improvements in HVLT-R Recognition (p=0.039), OTMT A (p=0.003), and OTMT B test of executive function (p=0.032). Conclusion(s): Neurocognitive scores in PWH were independent of COVID status, suggesting that higher frequencies of post-COVID neurocognitive dysfunction in PWH compared to HIV-seronegative people are due to HIV-associated factors more so than COVID. HIV-seronegative, post-COVID people demonstrate diminished recognition memory, processing speed, and executive function at 1 month post-COVID that improves by 4 months. Post-COVID neurocognitive dysfunction is present, if temporary, even in a highly vaccinated cohort of people.
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Background: SARS-CoV-2 continues to change over time due to genetic mutations and viral recombination.1 Given the changing landscape of COVID-19 variants and availability of COVID-19 vaccinations, disease severity during acute infection has also been variable. However, most research related to COVID-19 to date has not focused on evaluating differences in outcomes by the dominant variant and the impact it might have on post-acute sequalae of COVID-19 (PASC). Method(s): We developed a data mart of electronic health record data pertaining to COVID-19 in a single North American metropolitan health system (RUSH University Medical Center). Patients were selected for analysis if they had at least one documented infection of COVID-19. Date ranges were established per dominant variant, and the date of diagnosis was matched to variant. Variants were determined by the most prominent variant of concern (VOC) circulating in the city of Chicago. Variants were categorized by the following by date ranges: Wildtype+D614G (3/7/20-3/20/21), Alpha (3/21/21-6/19/21), Delta (6/20/21-12/11/21), Omicron BA.1 (12/12/21-3/19/22), Omicron BA.2 (3/20/22- 6/18/22), and Omicron BA.4/BA.5 (6/19/22-present (9/30/22). Subsequent clinical outcomes were examined, including hospitalization, intensive care unit admission, or death. We characterized our sample by conducting descriptive statistics including frequency and percent of outcome by variant. Result(s): 44,499 patients were included in this analysis with 30.23% requiring hospitalization, 4.25% being admitted to intensive care unit (ICU), and 2.35% resulting in death. The greatest percentage of hospitalizations occurred with the Alpha variant at 41.88% (N=928), and the greatest percentage of ICU admissions (6.43%) and death (3.15%) occurred with the Delta variant. The latest Omicron variant (Wave 6) showed an increase in hospitalizations (35.18%), as compared to early Omicron waves (Wave 4 and 5) but maintained similar ICU rates. Death rates continued to decline during the Omicron waves (Table 1). Conclusion(s): Although Alpha and Delta variants seem to have more severe outcomes compared to other variants, it is important to note that COVID-19 prevention, treatment access, and management continues to change, potentially influencing how outcomes may differ over time. Future work should determine factors to adjust for when examining variant-level differences.
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Background: HIV is a risk factor for severe acute COVID-19, but it is unknown whether HIV is a risk factor for long COVID. Method(s): We conducted a prospective observational cohort study of US adults with HIV (PWH) and HIV-seronegative adults with first SARS-CoV-2 infection within 4 weeks together with people who never had COVID-19. At enrollment, participants recalled the presence and severity of 49 long COVID-associated symptoms in the month prior to COVID-19. The same symptom survey was administered at 1, 2, 4, and 6 months post-COVID or post-enrollment for never- COVID participants. Post-COVID participants donated blood 1 and 4 months post-COVID, and never-COVID participants donated blood 0-1 times. Antibody titers to 18 coronavirus antigens and levels of 30 cytokines and hormones were quantified (Meso Scale Discovery). The Mann Whitney U test was used to compare continuous variables between groups, and Pearson's chi-squared test for categorical variables. Spearman correlation analyses were used to build networks of associations between cytokines and symptoms. Result(s): 341 participants enrolled between June 2021 and September 2022. Of these, 73 were PWH post-COVID, 121 were HIV-seronegative post-COVID, 78 were PWH never-COVID, and 69 were HIV-seronegative never-COVID. Over 85% of participants were vaccinated prior to COVID-19. Most participants with HIV were male sex at birth (83% post-COVID, 59% never-COVID), on ART ( >95%), with median CD4 counts >500. Over 60% of participants reported 1+ new or worsened symptoms 2-6 months post-COVID, with higher percentages in PWH at 2 months post-COVID (p< 0.05). PWH were more likely to report body ache, pain, confusion, memory problems, and thirst and had higher levels of creatine phosphokinase post-COVID than HIV-seronegative people. SARS-CoV-2 and non-SARS human coronavirus antibody titers did not differ between PWH and HIV-seronegative post-COVID participants. Cytokine associations with each other (network density) were significantly enriched at 1 month post-COVID in both PWH and HIV-seronegative people, with significantly less enrichment at 4 months post-COVID and in never- COVID participants. Levels of four analytes (cortisol, C5a, TGF-beta1, and TIM-3) associated with specific symptoms of long COVID. Conclusion(s): PWH may experience more symptoms post-COVID with a slightly different symptom profile than people without HIV. Inflammatory networks were active in PWH and people without HIV at 1 month post-COVID.
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Background Coronavirus disease 2019 (COVID-19) mortality remains high in those with cardiovascular disease (CVD). The temporal trend in higher COVID-19 mortality due to CVD has public health implications. We assessed the association between CVD and COVID-19 mortality throughout the COVID-19 pandemic. Methods We retrospectively studied all patients who received care for COVID-19 at Rush University System for Health during the pandemic (divided into 7 waves based on predominant virus variants and vaccine rollouts). CVD was defined as congestive heart failure (CHF), myocardial infarction (MI), cerebrovascular or peripheral vascular disease (ascertained by ICD codes). Using multivariable logistic regression, we assessed independent associations of COVID-19 mortality with age, sex, race, and 17 comorbidities in the Charlson comorbidity index, overall and stratified by pandemic waves. Results Of 43876 patients (mean age 40, 56% female, 14% with CVD), 1032 (2%) died from COVID-19 between March 2020 and August 2022. Adjusted for covariables, mortality was 3.2 times as likely in those with CVD as those without (OR=3.2, 95%CI 2.7-3.9;p<0.001). There was a trend toward increasing mortality associated with co-existing CVD as pandemic progressed to later waves (where Delta and Omicron were predominant), particularly in those with CHF or MI (Figure). Conclusion We found that COVID-19 mortality associated with co-existing CVD (particularly CHF and MI) increased temporally throughout the pandemic. [Formula presented]Copyright © 2023 American College of Cardiology Foundation
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Background. Which components of the immune response to SARS-CoV-2 vaccination best protect against subsequent infection remains unclear. We explored SARS-CoV-2 specific antibody and B-cell responses post 3rd dose vaccine and their relationship to subsequent SARS-CoV-2 infection. Methods. In a multicentre prospective cohort, adult subjects provided samples before and 14 days (d14) post 3rd dose vaccine with Pfizer-BioNTech 162b2. At 18-22 weeks post vaccine, subjects self-reported SARS-CoV-2 infection (confirmed by PCR or antigen test). We used electrochemiluminescence assays to quantify antibodies to SARS-CoV-2 spike subunit 1 (S1), subunit 2 (S2) and receptor-binding domain (RBD) in plasma (reported inWHOIU/mL). In a subset of subjects, we assessed SARS-CoV-2 specific differentiated B-cell (plasma cell) and memory B-cell responses from peripheral blood mononuclear cells. Unstimulated plasma cells, and memory B cells stimulated with R848 and IL2, were seeded on plates coated with RBD or full Spike antigen and antigen-specific responses measured by ELISpot (Mabtech ELISpot, Sweden). We compared between group differences by Wilcoxon signed rank or Mann-Whitney tests. Data are median [IQR] unless specified. Results. Of 133 subjects (age 43 [32-50], 81.2% female (table 1), 77 subjects in the B-cell subgroup (table 2)), 47 (35.3%) reported SARS-CoV-2 infection post 3rd vaccine. Antibody titres, plasma cell and memory B-cell responses all increased significantly at d14 post 3rd vaccine (Table 1 & 2, all P< 0.001). Although d14 antibody titres did not differ in those with and without subsequent infection (table 1), those reporting subsequent infection had significantly lower d14 RBD-specific plasma cells and a lower proportion of RBD-specific memory B-cells (Figure 1a-b, both P< 0.05). Similar results were observed with full-spike-specific memory B-cell responses (Figure 1d). The differences persisted when the non-infected group was restricted only to those reporting a confirmed close contact (n=26). Conclusion. Infection following 3rd dose vaccine is associated with lower d14 circulating and memory B cell responses, but not antibody titres, suggesting B-cell responses better predict protection against subsequent SARS-CoV-2 infection.
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Background. Long COVID is a heterogenous condition. We previously demonstrated distinct phenotypes of long COVID, but the impact of later waves caused by SARS-CoV-2 variants on long COVID presentations has not been described. Methods. We selected individuals with ongoing symptoms > 4 weeks from PCR-confirmed COVID-19 in a multicentre, prospective cohort study. We used multiple correspondence analysis and hierarchical clustering on self-reported symptoms to identify symptom clusters, in individuals recruited during two periods;cohort 1 from March 2020 to April 2021, and cohort 2 from April 2021 to March 2022. We explored differences in symptoms by mapping acute infection to one of four COVID-19 waves in Ireland (table 1) as well as vaccination status, and used Chi2 test to compare symptoms frequencies. Results. Demographics are shown in Table 2. Cluster analysis of each cohort demonstrated 3 distinct clusters in both cohorts, which shared similar clinical characteristics;a musculoskeletal/pain symptom cluster, a cardiorespiratory cluster and a third less symptomatic cluster (Figure 1). While symptoms within clusters were similar across both periods, in the cardiorespiratory cluster, the frequency of palpitations decreased (56% vs 16%) and cough increased (14% vs 45%) between reporting periods (both P< 0.01). Furthermore, a greater proportion of palpitations were reported in those with COVID-19 from waves 1 and 2 (35% and 28%) compared to 3 and 4 (both 12%, P< 0.001), and a greater proportion of chest pain in waves 1, 2 and 4 compared to wave 3. There were no differences in other symptoms (Table 3). Additionally there were significantly less palpitations reported in those vaccinated at the time of review (17% vs 31% P=0.002), but not chest pain (30% vs 39% P=0.13). In multivariate analysis, infection in wave 3 and 4 but not vaccination status remained significantly associated with lower reported palpitations (OR (95% CI) 0.28 (0.13-0.97) and 0.5 (0.06-0.87) for waves 3 and 4, both P< 0.05), and wave 3 infection remained independently associated with lower reported chest pain (OR 0.3 (0.25-0.7)). Conclusion. Three symptom clusters define long COVID across the two cohorts, but characteristics of the cardiorespiratory phenotype have evolved over time with evolution of SARS-CoV-2 variants. (Table Presented).
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Background: Coronavirus disease 2019 (COVID19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has varied clinical presentations from mild subclinical to severe disease with high mortality. Our aim was to determine whether examining immune-related gene expression early in infection could predict progression to severe disease. Methods: In subjects of the All Ireland Infectious Diseases Cohort study, we analysed expression of 579 genes with the NanoString nCounter Immunology panel in peripheral blood mononuclear cells in those with confirmed SARS-CoV-2 infection collected within 5 days of symptom onset and matched SARS-CoV-2 negative controls with respiratory infection. Subsequent maximum COVID19 disease severity was classified as mild or severe. Read counts were normalized using panel housekeeping genes. Expression changes in severity groups were estimated against control baseline. Results: Between April and July of 2020, we recruited 120 subjects, 62 with COVID19 and 58 controls, with average age 59 y.o. (IQR 34-88), 66% males and 69% Caucasian ethnicity. Maximal disease severity was used to separate COVID19 cases into mild (n=31) and severe (n=31). We identified 20 significantly deregulated genes between those with COVID19 and controls (;log2 fold;>0.5, p<0.05, Benjamin-Yekutieli p-adjustment). Function of 12 of these genes related to cytokine signaling, 9 upregulated genes to type I interferon signaling (MX1, IRF7, IFITM1, IFI35, STAT2, IRF4, PML, BST2, STAT1), while 7 downregulated genes mapped to innate immune function (IRF7, ICAM2, SERPING1, IFI16, BST2, FCER1A, PTK2). Expression in the severe group showed downregulation of FCER1A (innate immunity regulation), IL1B and TNF (inflammatory cytokines), and PTGS2 (inflammatory mediator) and greater upregulation of TNFSF4 (cytokine signaling) and PTK2 (innate immunity). Mild cases presented higher upregulation of IFIT2 (type I interferon signaling). Conclusion: Observed early downregulation of regulators and mediators of inflammation in those who developed severe COVID19, suggested dysregulation of inflammation. Specifically, IFIT2 upregulation in mild cases and FCER1A downregulation in severe cases, points to early differences in host responses centered on deregulation of the interferon and inflammation responses. Whether these patterns reflect delayed interferon involvement in pathways to control the infection and contribute to pathological inflammation and cytokine storms observed in severe COVID19 requires further research.
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Background: A disruption of the crosstalk between gut and lung has been implicated as a driver of severity during several respiratory-related diseases. Lung injury causes systemic inflammation, which disrupts gut barrier integrity, increasing the permeability to gut microbes and their products. This exacerbates inflammation, resulting in positive feedback. We applied a multi-omic systems biology approach to investigate the potential link between loss of gut barrier integrity and Coronavirus disease 2019 (COVID-19) severity. Methods: We analyzed plasma samples from age and gender-matched COVID-19 patients (n=60) with varying disease severity (mild, moderate, and severe) and 20 SARS-CoV2 negative controls. We measured markers and drivers of tight junction permeability and microbial translocation using ELISA;inflammation and immune activation markers using ELISA and multiplex cytokine arrays;untargeted metabolomic and lipidomic analyses using mass spectrometry;and plasma glycomes using capillary electrophoresis and lectin microarray. False discovery rate (FDR) was calculated to account for multiple comparisons. Results: Our data indicate, first, that severe COVID-19 is associated with a dramatic increase in the level of zonulin (FDR<0.00001), the only known physiological driver of intestinal tight junction permeability. This increased permeability associated with translocation of both bacterial (LPS binding protein (LBP) levels) and fungal (β-glucan levels) products into blood (FDR<0.01). The degree of intestinal permeability and microbial translocation strongly correlated with increased systemic inflammation (correlations with IL-6 and other inflammatory cytokines and markers) (FDR>0.05). Second, levels of metabolomic and lipidomic markers of gut and gut microbiota functionality including citrulline (a marker of healthy gut;decreased), succinic acid, and tryptophan catabolism metabolites (markers of microbial dysbiosis;increased) were disrupted during severe COVID-19 (FDR<0.05). Finally, the gut microbiome is known to release enzymes that degrade plasma glycans, which regulate inflammation and complement activation. Indeed, severe COVID-19 was associated with loss of the anti-complement activation galactosylated glycans from plasma and IgG glycoproteins (FDR<0.05). Conclusion: Our data provide multiple layers of evidence that a previously unappreciated factor with significant clinical implications, disruption in gut barrier integrity, is a potential force that contributes to COVID-19 severity.