Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups.

Introduction: While complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood. Methods: We therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome. Results: We show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID. Conclusion: In conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted.

Infection with SARS-CoV-2 Is Associated with Elevated Levels of IP-10, MCP-1, and IL-13 in Sepsis Patients.

Immunothrombosis, an excessive inflammatory response with simultaneous overactivation of the coagulation system, is a central pathomechanism in sepsis and COVID-19. It is associated with cellular activation, vascular damage, and microvascular thrombosis, which can lead to multiple organ failure and death. Here, we characterized factors related to immunothrombosis in plasma samples from 78 sepsis patients. In the course of routine clinical testing, SARS-CoV-2 was detected in 14 of these patients. Viral infection was associated with a higher mortality. Both, COVID-19 negative and COVID-19 positive sepsis patients showed increased levels of effectors of immunothrombosis, including platelet factor 4, D-dimer, nucleosomes, citrullinated histone H3, high mobility group box-1 protein, as well as phosphatidylserine-expressing platelet-derived extracellular vesicles, compared to healthy controls (n = 25). Using a 27-plex cytokine bead array, we found that Interleukin (IL)-1ra, IL-6, IL-8, IL-13, tumor necrosis factor (TNF)-α, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and granulocyte-colony stimulating factor (G-CSF) were elevated in both, COVID-19 negative and COVID-19 positive sepsis patients, as compared to healthy controls. SARS-CoV-2 infection was associated with elevated levels of IP-10, MCP-1, and IL-13, while all other mediators widely overlapped between COVID-19 negative and COVID-19 positive patients.

Regimen of Coronavirus Disease 2019 Vaccination Influences Extent and Kinetics of Antibody Avidity.

We investigated antibody titers and avidity after heterologous versus homologous coronavirus disease 2019 vaccination over 6 months after the second dose. We found a significantly higher avidity in regimens including at least 1 dose of the adenoviral vector vaccine ChAdOx1-S compared with 2 doses of the mRNA vaccine BNT162b2.

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

Indications that the Antimycotic Drug Amphotericin B Enhances the Impact of Platelets on Aspergillus.

Platelets are currently thought to harbor antimicrobial functions and might therefore play a crucial role in infections, e.g., those caused by Aspergillus or mucormycetes. The incidence of invasive fungal infections is increasing, particularly during the coronavirus disease 2019 (COVID-19) pandemic, and such infections continue to be life-threatening in immunocompromised patients. For this reason, the interaction of antimycotics with platelets is a key issue to evaluate modern therapeutic regimens. Amphotericin B (AmB) is widely used for the therapy of invasive fungal infections either as deoxycholate (AmB-D) or as a liposomal formulation (L-AmB). We showed that AmB strongly activates platelets within a few minutes. AmB concentrations commonly measured in the blood of patients were sufficient to stimulate platelets, indicating that this effect is highly relevant in vivo. The stimulating effect was corroborated by a broad spectrum of platelet activation parameters, including degranulation, aggregation, budding of microparticles, morphological changes, and enhanced adherence to fungal hyphae. Comparison between the deoxycholate and the liposomal formulation excluded the possibility that the liposomal part of L-Amb is responsible for these effects, as no difference was visible. The induction of platelet activation and alteration by L-AmB resulted in the activation of other parts of innate immunity, such as stimulation of the complement cascade and interaction with granulocytes. These mechanisms might substantially fuel the antifungal immune reaction in invasive mycoses. On the other hand, thrombosis and excessive inflammatory processes might occur via these mechanisms. Furthermore, the viability of L-AmB-activated platelets was consequently decreased, a process that might contribute to thrombocytopenia in patients.

Systemic Inflammation and Complement Activation Parameters Predict Clinical Outcome of Severe SARS-CoV-2 Infections.

Overactivation of the complement system has been characterized in severe COVID-19 cases. Complement components are known to trigger NETosis via the coagulation cascade and have also been reported in human tracheobronchial epithelial cells. In this longitudinal study, we investigated systemic and local complement activation and NETosis in COVID-19 patients that underwent mechanical ventilation. Results confirmed significantly higher baseline levels of serum C5a (24.5 ± 39.0 ng/mL) and TCC (11.03 ± 8.52 µg/mL) in patients compared to healthy controls (p < 0.01 and p < 0.0001, respectively). Furthermore, systemic NETosis was significantly augmented in patients (5.87 (±3.71) × 106 neutrophils/mL) compared to healthy controls (0.82 (±0.74) × 106 neutrophils/mL) (p < 0.0001). In tracheal fluid, baseline TCC levels but not C5a and NETosis, were significantly higher in patients. Kinetic studies of systemic complement activation revealed markedly higher levels of TCC and CRP in nonsurvivors compared to survivors. In contrast, kinetic studies showed decreased local NETosis in tracheal fluid but comparable local complement activation in nonsurvivors compared to survivors. Systemic TCC and NETosis were significantly correlated with inflammation and coagulation markers. We propose that a ratio comprising systemic inflammation, complement activation, and chest X-ray score could be rendered as a predictive parameter of patient outcome in severe SARS-CoV-2 infections.

Marked Increase in Avidity of SARS-CoV-2 Antibodies 7-8 Months After Infection Is Not Diminished in Old Age.

The kinetics of immunoglobulin G (IgG) avidity maturation during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection obtained from 217 participants of the Ischgl cohort, Austria, was studied 0.5-1.5 months (baseline) and 7-8 months (follow-up) after infection. The IgG avidity assay, using a modified IgG enzyme-linked immunosorbent assay (ELISA) and 5.5 M urea, revealed that old age does not diminish the increase in avidity, detected in all participants positive at both time points, from 18% to 42%. High avidity was associated with a marked residual neutralization capacity in 97.2.% of participants (211/217), which was even higher in the older age group, revealing an important role of avidity assays as easy and cheap surrogate tests for assessing the maturation of the immune system conveying potential protection against further SARS-CoV-2 infections without necessitating expensive and laborious neutralization assays.

Persistence of immunity to SARS-CoV-2 over time in the ski resort Ischgl.

Background In early March 2020, a SARS-CoV-2 outbreak in the ski resort Ischgl in Austria triggered the spread of SARS-CoV-2 throughout Austria and Northern Europe. In a previous study, we found that the seroprevalence in the adult population of Ischgl had reached 45% by the end of April, representing an exceptionally high level of local seropositivity in Europe. We performed a follow-up study in Ischgl, which is the first to show persistence of immunity and protection against SARS-CoV-2 and some of its variants at a community level. Methods Of the 1259 adults that participated in the baseline study, 801 have been included in the follow-up in November 2020. The study involved the analysis of binding and neutralizing antibodies and T cell responses. In addition, the incidence of SARS-CoV-2 and its variants in Ischgl was compared to the incidence in similar municipalities in Tyrol until April 2021. Findings For the 801 individuals that participated in both studies, the seroprevalence declined from 51.4% (95% confidence interval (CI) 47.9-54.9) to 45.4% (95% CI 42.0-49.0). Median antibody concentrations dropped considerably (5.345, 95% CI 4.833 - 6.123 to 2.298, 95% CI 2.141 - 2.527) but antibody avidity increased (17.02, 95% CI 16.49 - 17.94 to 42.46, 95% CI 41.06 - 46.26). Only one person had lost detectable antibodies and T cell responses. In parallel to this persistent immunity, we observed that Ischgl was relatively spared, compared to similar municipalities, from the prominent second COVID-19 wave that hit Austria in November 2020. In addition, we used sequencing data to show that the local immunity acquired from wild-type infections also helped to curb infections from variants of SARS-CoV-2 which spread in Austria since January 2021. Interpretation The relatively high level of seroprevalence (40-45%) in Ischgl persisted and might have been associated with the observed protection of Ischgl residents against virus infection during the second COVID-19 wave as well as against variant spread in 2021. Funding Funding was provided by the government of Tyrol and the FWF Austrian Science Fund.

Lipoprotein(a) and SARS-CoV-2 infections: Susceptibility to infections, ischemic heart disease and thromboembolic events.

BACKGROUND: Comorbidities including ischemic heart disease (IHD) worsen outcomes after SARS-CoV-2 infections. High lipoprotein(a) [Lp(a)] concentrations are a strong risk factor for IHD and possibly for thromboembolic events. We therefore evaluated whether SARS-CoV-2 infections modify the risk of high Lp(a) concentrations for IHD or thromboembolic events during the first 8.5 months follow-up of the pandemic. METHOD: Cohort study using data from the UK Biobank during the SARS-CoV-2 pandemic. Baseline Lp(a) was compared between SARS-CoV-2 positive patients and the population controls. RESULTS: SARS-CoV-2 positive patients had Lp(a) concentrations similar to the population controls. The risk for IHD increased with higher Lp(a) concentrations in both, the population controls (n = 435,104) and SARS-CoV-2 positive patients (n = 6937). The causality of the findings was supported by a genetic risk score for Lp(a). A SARS-CoV-2 infection modified the association with a steeper increase in risk for infected patients (interaction p-value = 0.03). Although SARS-CoV-2 positive patients had a five-times higher frequency of thromboembolic events compared to the population controls (1.53% vs. 0.31%), the risk was not influenced by Lp(a). CONCLUSIONS: SARS-CoV-2 infections enforce the association between high Lp(a) and IHD but the risk for thromboembolic events is not influenced by Lp(a).