The systemic renin-angiotensin system in COVID-19.

SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the "alternative" (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1-7. Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I-II) and alt-RAS (angiotensins 1-7 and 1-5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1-7. Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (angiotensin I: 86 versus 30 pmol/L, p < 0.01; angiotensin II: 114 versus 58 pmol/L, p < 0.05), demonstrating activation of classical RAS. The difference disappeared with analysis limited to patients not taking a RAS inhibitor (angiotensin I: 40 versus 31 pmol/L, p = 0.251; angiotensin II: 76 versus 99 pmol/L, p = 0.833). ALT-S in severe COVID-19 increased with time (days 1-6: 0.12; days 11-16: 0.22) and correlated with ACE2 concentration (r = 0.831). ACE-S was lower in severe versus non-severe COVID-19 (1.6 versus 2.6; p < 0.001), but ACE concentrations were similar between groups and correlated weakly with ACE-S (r = 0.232). ACE2 and ACE-S trajectories in severe COVID-19, however, did not differ between survivors and non-survivors. Overall RAS alteration in severe COVID-19 resembled severity of disease-matched patients with influenza. In mixed linear models, renin activity most strongly predicted angiotensin II and 1-7 levels. ACE2 also predicted angiotensin 1-7 levels and ALT-S. No single factor or the combined model, however, could fully explain ACE-S. ACE2 and ACE-S trajectories in severe COVID-19 did not differ between survivors and non-survivors. In conclusion, angiotensin II was elevated in severe COVID-19 but was markedly influenced by RAS inhibitors and driven by overall RAS activation. ACE-S was significantly lower with severe COVID-19 and did not correlate with ACE concentrations. A shift to the alt-RAS axis because of increased ACE2 could partially explain the relative reduction in angiotensin II levels.

Stopping of Mycophenolic Acid in Kidney Transplant Recipients for 2 Weeks Peri-Vaccination Does Not Increase Response to SARS-CoV-2 Vaccination-A Non-randomized, Controlled Pilot Study.

Introduction: Kidney transplant recipients (KTR) are at high risk of developing severe COVID-19. However, vaccine response in this population is severely impaired with humoral response rates of 36-54 and 55-69% after two or three doses of SARS-COV-2 vaccines, respectively. Triple immunosuppression and specifically the use of anti-proliferative agents such as mycophenolic acid (MPA) or azathioprine (AZA) have been identified as risk factors for vaccine hypo-responsiveness. Methods: We hypothesized that in vaccine non-responders to at least three previous vaccine doses, pausing of MPA or AZA for 1 week before and 1 week after an additional vaccination would improve humoral response rates. We conducted an open-label, non-randomized controlled pilot study including 40 KTR with no detectable humoral response after three or four previous vaccine doses. Primary endpoint was seroconversion following SARS-CoV-2 vaccination. MPA and AZA was paused in 18 patients 1 week before until 1 week after an additional vaccine dose while immunosuppression was continued in 22 patients. Results: There was no difference in the humoral response rate between the MPA/AZA pause group and the control group (29 vs. 32%, p > 0.99). Absolute antibody levels were also not statistically significantly different between the two groups (p = 0.716).Renal function in the MPA/AZA pause group remained stable and there was no detection of new onset donor-specific antibodies or an increase of donor-derived cell-free DNA serving as a marker of allograft damage throughout the study period. Conclusion: Pausing of MPA/AZA for 2 weeks peri-vaccination did not increase the rate of seroconversion in kidney transplant. However, one in three KTR without humoral immune response to at least three previous vaccinations developed antibodies after an additional vaccine dose supporting continued vaccination in non-responders.

Corticosteroid Treatment Prevents Lipopolysaccharide-Induced Increase of ACE2 and Reduces Fibrin Degradation Products in Bronchoalveolar Lavage Fluid.

The assessment of systemic corticosteroid effects on intrapulmonary disease biomarkers is challenging. This retrospective evaluation of a human endotoxemia model quantified ACE2 and fibrin degradation product (FDP) concentrations in bronchoalveolar lavage fluid (BALF) samples from a randomized, double-blind, placebo-controlled study (NCT01714427). Twenty-four healthy volunteers received either 2 × 40 mg intravenous dexamethasone or placebo. These doses were administered 12 h apart prior to bronchoscopy-guided intrabronchial lipopolysaccharide (LPS) stimulation (control: saline into the contralateral lung segment). We quantified ACE2 concentration, the Angiotensin-II-to-Angiotensin-1-7 conversion rate as well as FDP in BALF 6 h after LPS instillation. In placebo-treated subjects, LPS instillation increased ACE2 concentrations compared to unstimulated lung segments [1,481 (IQR: 736-1,965) vs. 546 (413-988) pg/mL; p = 0.016]. Dexamethasone abolished the increase in ACE2 concentrations (p=0.13). Accordingly, LPS instillation increased the Angiotensin-II-to-Angiotensin-1-7 conversion capacity significantly in the placebo cohort, indicating increased enzymatic activity (p = 0.012). FDP increased following LPS-instillation [8.9 (2.7-12.2) vs. 6.6 (0.9-9.6) ng/mL, p = 0.025] in the placebo group, while dexamethasone caused a shut-down of fibrinolysis in both lung segments. LPS instillation increased ACE2 concentration, its enzymatic activity and FDP, which was mitigated by systemic dexamethasone treatment. Our results strengthen previously published findings regarding the efficiency of corticosteroids for the treatment of COVID-19-induced acute lung injury.

Comparison of SARS-CoV-2 Antibody Response 4 Weeks After Homologous vs Heterologous Third Vaccine Dose in Kidney Transplant Recipients: A Randomized Clinical Trial.

Importance: Fewer than 50% of kidney transplant recipients (KTRs) develop antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Preliminary data suggest that a heterologous vaccination, combining mRNA and viral vector vaccines, may increase immunogenicity. Objective: To assess the effectiveness of a third dose of an mRNA vs a vector vaccine in KTRs who did not have antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Design, Setting, and Participants: This was a single center, single-blinded, 1:1 randomized clinical trial of a third dose of vaccine against SARS-CoV-2, conducted from June 15 to August 16, 2021, in 201 KTRs who had not developed SARS-CoV-2 spike protein antibodies after 2 doses of an mRNA vaccine. Data analyses were performed from August 17 to August 31, 2021. Interventions: mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as a third dose of a SARS-CoV-2 vaccine. Main Outcomes and Measures: The primary study end point was seroconversion after 4 weeks (29-42 days) following the third vaccine dose. Secondary end points included neutralizing antibodies and T-cell response assessed by interferon-γ release assays (IGRA). In addition, the association of patient characteristics and vaccine response was assessed using logistic regression, and the reactogenicity of the vaccines was compared. Results: Among the study population of 197 kidney transplant recipients (mean [SD] age, 61.2 [12.4] years; 82 [42%] women), 39% developed SARS-CoV-2 antibodies after the third vaccine. There was no statistically significant difference between groups, with an antibody response rate of 35% and 42% for the mRNA and vector vaccines, respectively. Only 22% of seroconverted patients had neutralizing antibodies. Similarly, T-cell response assessed by IGRA was low with only 17 patients showing a positive response after the third vaccination. Receiving nontriple immunosuppression (odds ratio [OR], 3.59; 95% CI, 1.33-10.75), longer time after kidney transplant (OR, 1.44; 95% CI, 1.15-1.83, per doubling of years), and torque teno virus plasma levels (OR, 0.92; 95% CI, 0.88-0.96, per doubling of levels) were associated with vaccine response. The third dose of an mRNA vaccine was associated with a higher frequency of local pain at the injection site compared with the vector vaccine, while systemic symptoms were comparable between groups. Conclusions and Relevance: This randomized clinical trial found that 39% of KTRs without an immune response against SARS-CoV-2 after 2 doses of an mRNA vaccine developed antibodies against the SARS-CoV-2 spike protein 4 weeks after a third dose of an mRNA or a vector vaccine. The heterologous vaccination strategy with a vector-based vaccine was well tolerated and safe but not significantly better than the homologous mRNA-based strategy. Trial Registration: EudraCT Identifier: 2021-002927-39.

A Randomized Clinical Trial of Anti-IL-6 Antibody Clazakizumab in Late Antibody-Mediated Kidney Transplant Rejection.

BACKGROUND: Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. Blocking IL-6 has been proposed as a promising therapeutic strategy. METHODS: We performed a phase 2 randomized pilot trial to evaluate the safety (primary endpoint) and efficacy (secondary endpoint analysis) of the anti-IL-6 antibody clazakizumab in late ABMR. The trial included 20 kidney transplant recipients with donor-specific, antibody-positive ABMR ≥365 days post-transplantation. Patients were randomized 1:1 to receive 25 mg clazakizumab or placebo (4-weekly subcutaneous injections) for 12 weeks (part A), followed by a 40-week open-label extension (part B), during which time all participants received clazakizumab. RESULTS: Five (25%) patients under active treatment developed serious infectious events, and two (10%) developed diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab displayed significantly decreased donor-specific antibodies and, on prolonged treatment, modulated rejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a negative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), and resolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the mean eGFR decline during part A was slower with clazakizumab compared with placebo (-0.96; 95% confidence interval [95% CI], -1.96 to 0.03 versus -2.43; 95% CI, -3.40 to -1.46 ml/min per 1.73 m2 per month, respectively, P=0.04). During part B, the slope of eGFR decline for patients who were switched from placebo to clazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab. CONCLUSIONS: Although safety data indicate the need for careful patient selection and monitoring, our preliminary efficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.