The Defenders of the Alveolus Succumb in COVID-19 Pneumonia to SARS-CoV-2 and Necroptosis, Pyroptosis, and PANoptosis.

Alveolar type II (ATII) pneumocytes as defenders of the alveolus are critical to repairing lung injury. We investigated the ATII reparative response in coronavirus disease 2019 (COVID-19) pneumonia, because the initial proliferation of ATII cells in this reparative process should provide large numbers of target cells to amplify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus production and cytopathological effects to compromise lung repair. We show that both infected and uninfected ATII cells succumb to tumor necrosis factor-α (TNF)-induced necroptosis, Bruton tyrosine kinase (BTK)-induced pyroptosis, and a new PANoptotic hybrid form of inflammatory cell death mediated by a PANoptosomal latticework that generates distinctive COVID-19 pathologies in contiguous ATII cells. Identifying TNF and BTK as the initiators of programmed cell death and SARS-CoV-2 cytopathic effects provides a rationale for early antiviral treatment combined with inhibitors of TNF and BTK to preserve ATII cell populations, reduce programmed cell death and associated hyperinflammation, and restore functioning alveoli in COVID-19 pneumonia.

Boosting corrects a memory B cell defect in SARS-CoV-2 mRNA-vaccinated patients with inflammatory bowel disease.

Immunosuppressed patients with inflammatory bowel disease (IBD) generate lower amounts of SARS-CoV-2 spike antibodies after mRNA vaccination than healthy controls. We assessed SARS-CoV-2 spike S1 receptor binding domain-specific (S1-RBD-specific) B lymphocytes to identify the underlying cellular defects. Patients with IBD produced fewer anti-S1-RBD antibody-secreting B cells than controls after the first mRNA vaccination and lower amounts of total and neutralizing antibodies after the second. S1-RBD-specific memory B cells were generated to the same degree in IBD and control groups and were numerically stable for 5 months. However, the memory B cells in patients with IBD had a lower S1-RBD-binding capacity than those in controls, which is indicative of a defect in antibody affinity maturation. Administration of a third shot to patients with IBD elevated serum antibodies and generated memory B cells with a normal antigen-binding capacity. These results show that patients with IBD have defects in the formation of antibody-secreting B cells and affinity-matured memory B cells that are corrected by a third vaccination.

Efficacy of Losartan in Hospitalized Patients With COVID-19-Induced Lung Injury: A Randomized Clinical Trial.

Importance: SARS-CoV-2 viral entry may disrupt angiotensin II (AII) homeostasis, contributing to COVID-19 induced lung injury. AII type 1 receptor blockade mitigates lung injury in preclinical models, although data in humans with COVID-19 remain mixed. Objective: To test the efficacy of losartan to reduce lung injury in hospitalized patients with COVID-19. Design, Setting, and Participants: This blinded, placebo-controlled randomized clinical trial was conducted in 13 hospitals in the United States from April 2020 to February 2021. Hospitalized patients with COVID-19 and a respiratory sequential organ failure assessment score of at least 1 and not already using a renin-angiotensin-aldosterone system (RAAS) inhibitor were eligible for participation. Data were analyzed from April 19 to August 24, 2021. Interventions: Losartan 50 mg orally twice daily vs equivalent placebo for 10 days or until hospital discharge. Main Outcomes and Measures: The primary outcome was the imputed arterial partial pressure of oxygen to fraction of inspired oxygen (Pao2:Fio2) ratio at 7 days. Secondary outcomes included ordinal COVID-19 severity; days without supplemental o2, ventilation, or vasopressors; and mortality. Losartan pharmacokinetics and RAAS components (AII, angiotensin-[1-7] and angiotensin-converting enzymes 1 and 2)] were measured in a subgroup of participants. Results: A total of 205 participants (mean [SD] age, 55.2 [15.7] years; 123 [60.0%] men) were randomized, with 101 participants assigned to losartan and 104 participants assigned to placebo. Compared with placebo, losartan did not significantly affect Pao2:Fio2 ratio at 7 days (difference, -24.8 [95%, -55.6 to 6.1]; P = .12). Compared with placebo, losartan did not improve any secondary clinical outcomes and led to fewer vasopressor-free days than placebo (median [IQR], 9.4 [9.1-9.8] vasopressor-free days vs 8.7 [8.2-9.3] vasopressor-free days). Conclusions and Relevance: This randomized clinical trial found that initiation of orally administered losartan to hospitalized patients with COVID-19 and acute lung injury did not improve Pao2:Fio2 ratio at 7 days. These data may have implications for ongoing clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04312009.

A novel wastewater-based epidemiology indexing method predicts SARS-CoV-2 disease prevalence across treatment facilities in metropolitan and regional populations.

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin's Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin's Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF's) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102-1.2 × 109 genome copies/L). Lag analysis of trends in Melvin's Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15-17 days at the statewide level and by up to 25 days at the regional/county level. Melvin's Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.

High-affinity memory B cells induced by SARS-CoV-2 infection produce more plasmablasts and atypical memory B cells than those primed by mRNA vaccines.

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A+ subset and secondary MBCs that are mostly IgG+ and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses.

A multi-center phase II randomized clinical trial of losartan on symptomatic outpatients with COVID-19.

BACKGROUND: The SARS-CoV-2 virus enters cells via Angiotensin-converting enzyme 2 (ACE2), disrupting the renin-angiotensin-aldosterone axis, potentially contributing to lung injury. Treatment with angiotensin receptor blockers (ARBs), such as losartan, may mitigate these effects, though induction of ACE2 could increase viral entry, replication, and worsen disease. METHODS: This study represents a placebo-controlled blinded randomized clinical trial (RCT) to test the efficacy of losartan on outpatients with COVID-19 across three hospital systems with numerous community sites in Minnesota, U.S. Participants included symptomatic outpatients with COVID-19 not already taking ACE-inhibitors or ARBs, enrolled within 7 days of symptom onset. Patients were randomized to 1:1 losartan (25 mg orally twice daily unless estimated glomerular filtration rate, eGFR, was reduced, when dosing was reduced to once daily) versus placebo for 10 days, and all patients and outcome assesors were blinded. The primary outcome was all-cause hospitalization within 15 days. Secondary outcomes included functional status, dyspnea, temperature, and viral load. (clinicatrials.gov, NCT04311177, closed to new participants). FINDINGS: From April to November 2020, 117 participants were randomized 58 to losartan and 59 to placebo, and all were analyzed under intent to treat principles. The primary outcome did not differ significantly between the two arms based on Barnard's test [losartan arm: 3 events (5.2% 95% CI 1.1, 14.4%) versus placebo arm: 1 event (1.7%; 95% CI 0.0, 9.1%)]; proportion difference -3.5% (95% CI -13.2, 4.8%); p = 0.32]. Viral loads were not statistically different between treatment groups at any time point. Adverse events per 10 patient days did not differ signifcantly [0.33 (95% CI 0.22-0.49) for losartan vs. 0.37 (95% CI 0.25-0.55) for placebo]. Due to a lower than expected hospitalization rate and low likelihood of a clinically important treatment effect, the trial was terminated early. INTERPRETATION: In this multicenter blinded RCT for outpatients with mild symptomatic COVID-19 disease, losartan did not reduce hospitalizations, though assessment was limited by low event rate. Importantly, viral load was not statistically affected by treatment. This study does not support initiation of losartan for low-risk outpatients.

Initial determination of COVID-19 seroprevalence among outpatients and healthcare workers in Minnesota using a novel SARS-CoV-2 total antibody ELISA.

OBJECTIVES: To avoid the significant risks posed by the use of COVID-19 serology tests with supply chain constraints or poor performance characteristics, we developed an in-house SARS-CoV-2 total antibody test. Our test was compared with three commercial methods, and was used to determine COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. METHODS: Seventy-nine plasma and serum samples from 50 patients 4-69 days after symptom onset who tested positive by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab were used to evaluate our test's clinical performance. Seropositive samples were analyzed for IgG titers in a follow-up assay. Thirty plasma and serum from 12 patients who tested negative by a SARS-CoV-2 PCR method using a nasopharyngeal (NP) swab and 210 negative pre-pandemic serum samples were also analyzed. Among samples from patients > 14 days after symptom onset, the assay had 100% clinical sensitivity and 100% clinical specificity, 100% positive predictive value and 100% negative predictive value. Analytical specificity was 99.8%, indicating minimal cross-reactivity. A screening study was conducted to ascertain COVID-19 seroprevalence among healthcare workers and outpatients in Minnesota. RESULTS: Analysis of serum collected between April 13 and May 21, 2020 indicated a COVID-19 seroprevalence of 2.96% among 1,282 healthcare workers and 4.46% among 2,379 outpatients. CONCLUSIONS: Our in-house SARS-CoV-2 total antibody test can be used to conduct reliable epidemiological studies to inform public health decisions during the COVID-19 pandemic.

Understanding the renin-angiotensin-aldosterone-SARS-CoV axis: a comprehensive review.

IMPORTANCE: Coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic with significant morbidity and mortality since first appearing in Wuhan, China, in late 2019. As many countries are grappling with the onset of their epidemics, pharmacotherapeutics remain lacking. The window of opportunity to mitigate downstream morbidity and mortality is narrow but remains open. The renin-angiotensin-aldosterone system (RAAS) is crucial to the homeostasis of both the cardiovascular and respiratory systems. Importantly, SARS-CoV-2 utilises and interrupts this pathway directly, which could be described as the renin-angiotensin-aldosterone-SARS-CoV (RAAS-SCoV) axis. There exists significant controversy and confusion surrounding how anti-hypertensive agents might function along this pathway. This review explores the current state of knowledge regarding the RAAS-SCoV axis (informed by prior studies of SARS-CoV), how this relates to our currently evolving pandemic, and how these insights might guide our next steps in an evidence-based manner. OBSERVATIONS: This review discusses the role of the RAAS-SCoV axis in acute lung injury and the effects, risks and benefits of pharmacological modification of this axis. There may be an opportunity to leverage the different aspects of RAAS inhibitors to mitigate indirect viral-induced lung injury. Concerns have been raised that such modulation might exacerbate the disease. While relevant preclinical, experimental models to date favour a protective effect of RAAS-SCoV axis inhibition on both lung injury and survival, clinical data related to the role of RAAS modulation in the setting of SARS-CoV-2 remain limited. CONCLUSION: Proposed interventions for SARS-CoV-2 predominantly focus on viral microbiology and aim to inhibit viral cellular injury. While these therapies are promising, immediate use may not be feasible, and the time window of their efficacy remains a major unanswered question. An alternative approach is the modulation of the specific downstream pathophysiological effects caused by the virus that lead to morbidity and mortality. We propose a preponderance of evidence that supports clinical equipoise regarding the efficacy of RAAS-based interventions, and the imminent need for a multisite randomised controlled clinical trial to evaluate the inhibition of the RAAS-SCoV axis on acute lung injury in COVID-19.