Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals

Background/Objectives: Emerging evidence suggests that complement system infection-dependent hyperactivation may worsen COVID-19 outcome. We investigated the role of predicted high impact variants -referred as Qualifying Variants (QVs) -of complement system genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. Method(s): Exploiting Whole-Exome Sequencing (WES) data and 56 complement system genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (age >= 60 y.o.) and 56,885 European individuals from the gnomAD database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized COVID-19 patients. Result(s): We found an enrichment of QVs in three genes (MASP1, COLEC10 and COLEC11), which belong to the lectin pathway, in the asymptomatic cohort. Moreover, individuals with QVs showed lower serum levels of Masp1 and of prothrombin activity compared to controls while no differences were observed for CH50 and AH50 levels that measure the activity of classical and alternative complement pathways, respectively. Finally, integrative analyses of genome-wide association study and expression quantitative loci traits data showed a correlation between polymorphisms associated with asymptomatic COVID-19 and decreased expression of MASP1, COLEC11 and COLEC10 genes in lung tissue. Conclusion(s): This study suggests that rare genetic variants can protect from severe COVID-19 by mitigating the activation of lectin pathway and prothrombin activity.

Application of urinary proteomics for biomarker discovery in respiratory diseases

Background: To search for molecular biomarkers of pulmonary pathologies using non-invasive samples, such as urine, is of high clinical relevance. However, there are almost no proteomic studies using urine applied to respiratory diseases. Aim(s): To develop a biomarker discovery strategy using non-targeted proteomics in urine with applicability to different pulmonary diseases. Method(s): Urine samples were centrifuged and DTT treated to decrease uromodulin (THP). Low-THP samples were concentrated (ultrafiltration), ultracentrifugated, and exosome free urine was analysed using LC-MS/MS. GO terms/Pathway analyses were performed using STRING database. Result(s): Urine proteome (765 proteins) was enriched (FDR < 0.05) in proteins from different tissues, including respiratory system (N = 124), lung (N = 107), and immune system (N = 88). We detected an enrichment of relevant pathways for respiratory diseases, including several innate (e.g., TLR and NFkB pathways, complement system), and adaptive (e.g., interleukin signalling) immune system pathways. Some of these proteins have been previously studied in respiratory system disease (e.g., MPO, NAPSA, CHL1, FREM2, PLG), lower respiratory tract disease (e.g., NCAM1, MTOR, SERPINA1), viral infectious disease (e.g., ITIH4, CD209, CLEC4M, CD55), or specific pathologies such as coronavirus infection (e.g., ACE2, TMPRSS2), bronchiectasis (e.g., SAA1, SAA2, ELANE) or asthma (e.g., IGFALS, IGFBP7, HSPG2, DPP4, CD44, IL6R, MASP1). Conclusion(s): We have developed a protocol for the detection of proteomic biomarkers in urine. This proteome is enriched in proteins from the immune and respiratory systems, with a potential clinical and translational relevance.

Labeled TEMPO-Oxidized Mannan Differentiates Binding Profiles within the Collectin Families.

Establishing the rapid and accurate diagnosis of sepsis is a key component to the improvement of clinical outcomes. The ability of analytical platforms to rapidly detect pathogen-associated molecular patterns (PAMP) in blood could provide a powerful host-independent biomarker of sepsis. A novel concept was investigated based on the idea that a pre-bound and fluorescent ligand could be released from lectins in contact with high-affinity ligands (such as PAMPs). To create fluorescent ligands with precise avidity, the kinetically followed TEMPO oxidation of yeast mannan and carbodiimide coupling were used. The chemical modifications led to decreases in avidity between mannan and human collectins, such as the mannan-binding lectin (MBL) and human surfactant protein D (SP-D), but not in porcine SP-D. Despite this effect, these fluorescent derivatives were captured by human lectins using highly concentrated solutions. The resulting fluorescent beads were exposed to different solutions, and the results showed that displacements occur in contact with higher affinity ligands, proving that two-stage competition processes can occur in collectin carbohydrate recognition mechanisms. Moreover, the fluorescence loss depends on the discrepancy between the respective avidities of the recognized ligand and the fluorescent mannan. Chemically modulated fluorescent ligands associated with a diversity of collectins may lead to the creation of diagnostic tools suitable for multiplex array assays and the identification of high-avidity ligands.

Detection of Aspergillus spp. and Candida albicans antigenemia in a sample of Iraqi patients with severe COVID-19

Introduction: Since December 2019, coronavirus disease 2019 (COVID-19), which is caused by SARSCoV- 2, has spread locally in Wuhan, China, and later on, a worldwide outbreak occurred. Invasive fungal infections can cause complications in critically ill immunocompromised patients of COVID-19, especially those admitted to intensive care units and who required mechanical ventilation. Candida albicans have been the most common pathogenic species, followed by other Candida spp. Mannan is a major component of the Candida cell wall and can be detected by the enzyme-linked immunosorbent assay (ELISA) in blood and other fluids. Invasive pulmonary aspergillosis is considered a lifethreatening infection, especially among immunocompromised patients. COVID-19-associated pulmonary aspergillosis has emerged as an important complication among patients in the intensive care units. Galactomannan (GM) is a major cell-wall component of Aspergillus spp. and can be found in body fluids. Blood GM can be detected by the enzyme immunoassay. The aim of the current study is to assess the frequency of aspergillosis and candidiasis among COVID-19 patients in some hospitals in Baghdad by using GM and mannan biomarkers. Methods: During the period from February 2020 to May 2021, 175 COVID-19 blood samples of patients were collected and a sandwich ELISA test was performed to detect GM Ag of Aspergillus spp. and mannan Ag of Candida spp. Results: Regarding C-reactive protein (CRP), significant differences were seen among Aspergillus/- COVID-19 patients ( p 0.029). Regarding sex and age group, the results indicated that of a total of 175 adult patients with positive COVID-19, more than half of the patients were males. Regarding the distribution of mannan Ag and GM Ag in COVID-19 patients, it was seen that out of the 175 patients, 167 (95.43%) Candida mannan Ag were negative and only 8 (4.57%) were positive, and 170 (79.14%) Aspergillus GM Ag were negative and only 5 (2.86%) were positive. It was also seen that 2 patients (1.14%) who had both Candida mannan and Aspergillus GM were positive and 173 (98.6%) were negative. No statistically significant difference was seen in candidiasis and aspergillosis among patients with COVID-19 regarding age group, sex, underlying chronic diseases (hypertension and diabetes mellitus), and biochemical tests. Conclusion: COVID-19 infections increased with age and were seen more in males than in females. The percentage of infection with C. albicans and Aspergillus spp. among COVID-19 patients was not significant, and this may come from the random collection of samples from patients with different stages of illness. A significant correlation was found between Aspergillus GM Ag in COVID-19 patients and the CRP test.

Identification of Complement-Related Missense Variants in Pediatric Patients with Acute and Post COVID-19 Syndromes

Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses. Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms. Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature. Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. 1. In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients);and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes. 2. The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation. Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. [Formula prese ted] Disclosures: Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy;Gilead Corporation: Honoraria;Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials;Sanofi: Other: clinical trials;Ocopeptides: Other: clinical trials;GSK: Other: clinical trials;Incyte: Other: clinical trials;Takeda: Other: clinical trials;Amgen: Other: clinical trials;Janssen: Other: clinical trials;novartis: Other: clinical trials;Celgene: Other: clinical trials;Roche: Other: clinical trials;Astellas: Other: clinical trials.