ELISA-Based Analysis Reveals an Anti-SARS-CoV-2 Protein Immune Response Profile Associated with Disease Severity

Since the start of the COVID-19 pandemic, many studies have investigated the humoral response to SARS-CoV-2 during infection. Studies with native viral proteins constitute a first-line approach to assessing the overall immune response, but small peptides are an accurate and valuable tool for the fine characterization of B-cell epitopes, despite the restriction of this approach to the determination of linear epitopes. In this study, we used ELISA and peptides covering a selection of structural and non-structural SARS-CoV-2 proteins to identify key epitopes eliciting a strong immune response that could serve as a biological signature of disease characteristics, such as severity, in particular. We used 213 plasma samples from a cohort of patients well-characterized clinically and biologically and followed for COVID-19 infection. We found that patients developing severe disease had higher titers of antibodies mapping to multiple specific epitopes than patients with mild to moderate disease. These data are potentially important as they could be used for immunological profiling to improve our knowledge of the quantitative and qualitative characteristics of the humoral response in relation to patient outcome.

Shift of lung macrophage composition is associated with COVID-19 disease severity and recovery

Though it has been 2 years since the start of the Coronavirus Disease 19 (COVID-19) pandemic, COVID-19 continues to be a worldwide health crisis. Despite the development of preventive vaccines, very little progress has been made to identify curative therapies to treat COVID-19 and other inflammatory diseases which remain a major unmet need in medicine. Our study sought to identify drivers of disease severity and death to develop tailored immunotherapy strategies to halt disease progression. Here we assembled the Mount Sinai COVID-19 Biobank which was comprised of ~600 hospitalized patients followed longitudinally during the peak of the pandemic. Moderate disease and survival were associated with a stronger antigen (Ag) presentation and effector T cell signature, while severe disease and death were associated with an altered Ag presentation signature, increased numbers of circulating inflammatory, immature myeloid cells, and extrafollicular activated B cells associated with autoantibody formation. Strikingly, we found that in severe COVID-19 patients, lung tissue resident alveolar macrophages (AM) were not only severely depleted, but also had an altered Ag presentation signature, and were replaced by inflammatory monocytes and monocyte-derived macrophages (MoMϕ). Notably, the size of the AM pool correlated with recovery or death, while AM loss and functionality were restored in patients that recovered. These data therefore suggest that local and systemic myeloid cell dysregulation is a driver of COVID-19 severity and that modulation of AM numbers and functionality in the lung may be a viable therapeutic strategy for the treatment of critical lung inflammatory illnesses.

Prevalence, characteristics, and predictors of Long COVID among diagnosed cases of COVID-19

Background: Long COVID or long-term complication after COVID-19 has the ability to affect health and quality of life. Knowledge about the burden and predictors could aid in their prevention and management. Most of the studies are from high-income countries and focus on severe cases. We did this study to estimate the prevalence and identify the characteristics and predictors of Long COVID among our patients. Methodology: We recruited adult (≥18 years) patients who were diagnosed as Reverse Transcription Polymerase Chain Reaction (RTPCR) confirmed SARS-COV-2 infection and were either hospitalized or tested on outpatient basis. Eligible participants were followed up telephonically after four weeks of diagnosis of SARS-COV-2 infection to collect data on sociodemographic, clinical history, vaccination history, Cycle threshold (Ct) values during diagnosis and other variables. Characteristic of Long COVID were elicited, and multivariable logistic regression was done to find the predictors of Long COVID. Results: We have analyzed 487 individual data with a median follow-up of 44 days (Inter quartile range (IQR): 39,47). Overall, Long COVID was reported by 29.2% (95% Confidence interval (CI): 25.3%,33.4%) participants. Prevalence of Long COVID among patients with mild/moderate disease (n = 415) was 23.4% (95% CI: 19.5%,27.7%) as compared to 62.5% (95% CI: 50.7%,73%) in severe/critical cases(n=72). The most common Long COVID symptom was fatigue (64.8%) followed by cough (32.4%). Statistically significant predictors of Long COVID were - Pre-existing medical conditions (Adjusted Odds ratio (aOR)=2.00, 95% CI: 1.16,3.44), having a more significant number of symptoms during acute phase of COVID-19 disease (aOR=11.24, 95% CI: 4.00,31.51), two doses of COVID-19 vaccination (aOR=2.32, 95% CI: 1.17,4.58), the severity of illness (aOR=5.71, 95% CI: 3.00,10.89) and being admitted to hospital (Odds ratio (OR)=3.89, 95% CI: 2.49,6.08). Conclusion: A considerable proportion of COVID-19 cases reported Long COVID symptoms. More research is needed in Long COVID to objectively assess the symptoms and find the biological and radiological markers.

Temperature Influences on Powdery Mildew Susceptibility and Development in the Hop Cultivar Cascade

The hop cultivar Cascade possesses partial resistance to powdery mildew (Podosphaera macularis) that can be overcome by recently emerged, virulent isolates of the fungus. Given that hop is a long-lived perennial and that brewers still demand Cascade, there is a need to better understand factors that influence the development of powdery mildew on this cultivar. Growth chamber experiments were conducted to quantify the effect of constant, transient, and fluctuating temperature on Cascade before, concurrent to, and after inoculation as contrasted with another powdery mildew susceptible cultivar, Symphony. Exposure of plants to supraoptimal temperature (26 and 32°C) prior to inoculation led to more rapid onset of ontogenic resistance in intermediately aged leaves in Cascade as compared to Symphony. Cascade was overall less susceptible to powdery mildew when exposed to constant temperature ranging from 18 to 32°C directly after inoculation. However, cultivar also interacted with temperature such that proportionately fewer and smaller colonies developed on Cascade than Symphony at supraoptimal yet permissive temperatures for disease. When plants were inoculated and then exposed to high temperature, colonies became progressively more tolerant to temperatures of 26 to 30°C with increasing time from inoculation to exposure, as moderated by cultivar, the specific temperature, and their interaction. Subjecting plants to simulated diurnal temperature regimes at the time of inoculation or 24 h later indicated Cascade and Symphony responded proportionately similar on days predicted to be marginally unfavorable or marginally favorable for powdery mildew, although Cascade was quantitatively less susceptible than Symphony. In sum, this research indicates that Cascade is overall less susceptible to powdery mildew than Symphony, and supraoptimal temperature before, concurrent to, or after infection may interact differentially to moderate disease risk in Cascade. Therefore, cultivar-specific risk assessments for powdery mildew appear warranted.

Longitudinal characterisation of phagocytic and neutralisation functions of anti-Spike antibodies in plasma of patients after SARS-CoV-2 infection.

Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions.

Comparison Of Retinopathy And Hearing Impairment In Poorly Controlled Diabetics In Population Of Muzaffarabad AJK

Aim: Prevalence and comparison of retinopathy and sensorineural hearing loss in poorly managed diabetics Methodology: A cross sectional study was designed with the approval of the Ethical Review Board Committee. The study enrolled 50 people who met the inclusion criteria. Anthropometric data were collected using standardized procedures. All laboratory assays were performed on the initial visit to the diabetes research center's laboratory. Tuning fork and PTA examinations of the ears were performed. The scores of Rinne and Weber were recorded and synchronised. Fundoscopy was used in ophthalmic investigations. Results: The Rinne test was found to be normal in 50(100%) cases of population while Weber’s test was centralized in 43(86%) of patients, 1(2%) case was lateralized to right, 5(10%)cases were lateralized to left, 1(2%) case had moderate disease. PTA scores revealed mild disease in 1(2%), moderate 14(28%), severe 1(2%) and 34(68%) patients showed normal results. Fundoscopy demonstrated normal results in 11(22%), Grade 1 retinopathy in 15(30%), grade 2 RP in 17(34%) and grade 3 RP in 7(14%). Conclusion: Retinopathy is more prevalent with poorly managed diabetic individuals in contrast to hearing impairment. Key words: Retinopathy, Fundoscopy, Diabetes mellitus

Single cell gene expression profiling of nasal ciliated cells reveals distinctive biological processes related to epigenetic mechanisms in patients with severe COVID-19

Objective: To explore the molecular processes associated with cellular regulatory programs in patients with COVID-19, including gene activation or repression mediated by epigenetic mechanisms. We hypothesized that a comprehensive gene expression profiling of nasopharyngeal epithelial cells might expand understanding of the pathogenic mechanisms of severe COVID-19. Methods: We used single-cell RNA sequencing (scRNAseq) profiling of ciliated cells (n = 12725) from healthy controls (SARS-CoV-2 negative n =13) and patients with mild/moderate (n =13) and severe (n =14) COVID-19. ScRNAseq data at the patient level were used to perform gene set and pathway enrichment analyses. We prioritized candidate miRNA-target interactions and epigenetic mechanisms. Results: Pathways linked to mitochondrial function, misfolded proteins, and membrane permeability were upregulated in patients with mild/moderate disease compared to healthy controls. Besides, we noted that compared to mild/moderate disease, cells derived from severe COVID-19 patients had downregulation of sub-networks associated with epigenetic mechanisms, including DNA and histone H3K4 methylation and chromatin remodeling regulation. We found 11-ranked miRNAs that may explain miRNA-dependent regulation of histone methylation, some of which share seed sequences with SARS-CoV-2 miRNAs. Conclusion: Our results may provide novel insights into the epigenetic mechanisms mediating the clinical course of SARS-CoV-2 infection.

Pharmacokinetics of ß-d-N4-hydroxycytidine, the active metabolite of prodrug molnupiravir, in non-plasma compartments of patients with SARS-CoV-2 infection

ABSTRACTBackgroundMolnupiravir, an orally administered prodrug of the broadly active, direct-acting antiviral, ribonucleoside analogue ß-d-N4-hydroxycytidine (NHC) is a promising COVID-19 drug candidate. We characterised the pharmacokinetics of NHC in saliva, nasal secretions and tears of patients enrolled in the phase I AGILE trial (NCT04746183) to understand its potential in preventing infection and transmission.MethodsPatients with PCR-confirmed SARS-CoV-2 infection, within 5 days of symptom onset with mild-to-moderate disease were randomised to oral molnupiravir 300, 600 or 800 mg twice daily or placebo. Plasma and non-plasma (saliva, nasal secretions and tears) samples were collected at pre-dose, 0.5, 1, 2, and 4 hours post-dose on study days 1 and 5 and molnupiravir and NHC measured by LC/MS with a lower limit of quantification of 2.5 ng/mL in all matrices. Pharmacokinetic parameters were determined by noncompartmental methods and non-plasma:plasma ratios (RNP:P; based on AUC0-4) calculated.ResultsTwelve participants (n=4 per dosing arm; 75% female) completed the study. NHC Tmax ranged between 1.00-4.00 hours for saliva (n=21) and nasal swabs (n=22) and 0.50-4.00 hours (n=17) for tears compared to 1.00-2.00 hours for plasma (n=19). Median (range) saliva RNP:P pooled across doses was 0.03 (0.01-0.11); n=16. RNP:P for nasal secretions and tears were 0.21 (0.05-0.73); n=17 and 0.22 (0.09-1.05); n=12, respectively. Non-plasma and plasma concentrations were significantly correlated (p<0.0001).ConclusionThese data provide encouraging information regarding the distribution of NHC at sites of viral transmission and have important implications for prophylactic coverage.

Adjunctive Techniques for Repair of Ischaemic Mitral Regurgitation

Ischaemic mitral regurgitation is a complex process with debate in the literature as to the optimal treatment pathway. Multiple therapies are available to alleviate mitral regurgitation including medical management, transcatheter edge-to-edge repair, mitral valve repair and mitral valve replacement. Medical management with goal-directed therapy should be utilised in patients with heart failure and mild-to-moderate regurgitation. Transcatheter approaches are typically used in patients with prohibitive operative risk, although their use is expanding, especially in those with functional mitral regurgitation who are not responding to goal-directed medical therapy. It is generally accepted that patients with mild-to-moderate disease can avoid valve intervention if successful revascularisation is performed. A higher consideration should be given to valve replacement over repair in patients with severe mitral regurgitation in the setting of myocardial ischaemia. Operative course must be personalised to each patient, and continues to develop with improving technologies and ongoing research into optimal treatment.

Monoclonal Antibodies against SARS-CoV-2: Current Scenario and Future Perspectives

Monoclonal antibodies (mAbs) have been known since the 1970s. However, their therapeutic potential in the medical field has recently emerged, with the advancement of manufacturing techniques. Initially exploited mainly in the oncology field, mAbs have become increasingly relevant in Infectious Diseases. Numerous mAbs have been developed against SARS-CoV 2 and have proven their effectiveness, especially in the management of the mild-to-moderate disease. In this review, we describe the monoclonal antibodies currently authorized for the treatment of the coronavirus disease 19 (COVID-19) and offer an insight into the clinical trials that led to their approval. We discuss the mechanisms of action and methods of administration as well as the prophylactic and therapeutic labelled indications (both in outpatient and hospital settings). Furthermore, we address the critical issues regarding mAbs, focusing on their effectiveness against the variants of concern (VoC) and their role now that a large part of the population has been vaccinated. The purpose is to offer the clinician an up-to-date overview of a therapeutic tool that could prove decisive in treating patients at high risk of progression to severe disease.