SARS-CoV-2 Infection Increases the Gene Expression Profile for Alzheimer's Disease Risk

The COVID-19 pandemic is a global outbreak of coronavirus, an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One in five adults who have had COVID-19 in the past was still experiencing any one of the symptoms of long COVID like headache, brain fog, fatigue, and shortness of breath. Up to 30% of individuals with mild to severe infection show diverse neurological symptoms, including dementias. Hence, it is very much important to characterize the neurotropism and neurovirulence of the SARS-CoV-2 virus. This helps us understand the mechanisms involved in initiating inflammation in the brain, further leading to the development of earlyonset Alzheimer's disease and related dementias (ADRDs). In our brain gene expression analysis, we found that severe COVID-19 patients showed increased expression of innate immune response genes and genes that are implicated in AD pathogenesis. To study the infection-induced ADRDs, we used a mouse-adapted strain of the SARS-CoV-2 (MA10) virus to infect mice of different age groups (3, 6, and 20 Months). In this study, we found that aged mice showed evidence of viral neurotropism, prolonged viral infection, increased expression of tau aggregator FKBP51, interferoninducible gene Ifi204, and complement genes like C4 and C5AR1. Brain histopathology also showed the AD signature including tau-phosphorylation, tau-oligomerization, and alpha-synuclein expression in aged MA10-infected mice. The results from gene expression profiling of SARS-CoV-2 infected and AD brains and studies with MA10 aged mice show that COVID-19 infection increases the risk of AD in the aged population. Furthermore, this study helps us to understand the crucial molecular markers that are regulated during COVID infection that could act as major players in developing ADRDs. Future studies will be involved in understanding the molecular mechanisms of ADRD in response to COVID infection and developing novel therapies targeting AD.

Regional effects on efficacy and safety of vilobelimab in mechanically ventilated patients with severe COVID-19

Introduction: Poor outcomes in COVID-19 patients (pt) are associated with C5a-C5aR axis activation. A C5a-specific monoclonal antibody, vilobelimab (VILO), improves outcomes in critically ill COVID-19 pt in a Phase 3 randomized, double-blind, placebo (PLC)- controlled study [1]. Method(s): COVID-19 pt within 48 h of intubation were randomly assigned to receive 6, 800 mg infusions of VILO or PLC at a 1:1 ratio on top of standard of care. Predefined subgroup analyses by region and country were performed. Result(s): Forty-six (46) hospitals on 4 continents randomized 369 pt: VILO (n = 178), PLC (n = 191). VILO significantly reduced 28- (HR 0.67;95% CI 0.48-0.96;p = 0.027) and 60-Day mortality (HR 0.67;95% CI 0.48-0.93, p = 0.0163) using a predefined, unstratified per protocol analysis. Mortality rates at 28- and 60-days and VILO treatment effects, however, differed substantially between regions: Western Europe HR for 60-day mortality 0.59 [0.37-0.95], South Africa plus Russian Federation HR 0.62 [0.28-1.38] and South America HR 0.80 [0.46-1.39] (Fig. 1). The weak signal in South America is predominately driven by Brazil (n = 74), which showed a significant age imbalance with a median 9-years younger PLC group (44.5-years-old vs 53.5-years-old) with low 60-day mortality of ~ 32.5% in the PLC group versus ~ 43.3% in Western Europe. Adjusting for age group categories (<= 30, 31-40, 41-50, 51-60, > 60;Cox regression) for 60-day mortality changed the HR in Brazil (0.96 [0.44-2.10] for continuous age-adjustment) to values near the estimate for the entire study population (HR 0.77 [0.35-1.69] for age in categories), suggesting a by chance imbalance and not a statistically evident weaker effect in Brazil. Conclusion(s): Regional efficacy differences between the rest of the world and South America were driven by age imbalances between treatment groups, which do not diminish the robust efficacy signal for VILO in severe COVID-19.

Pharmacokinetic/pharmacodynamic analysis of vilobelimab and anaphylatoxin c5a as well as antidrug antibodies in Panamo: a phase 3 study in critically ill, mechanically ventilated COVID-19 patients

Introduction: C5a-C5aR axis activation is associated with increased mortality in severe COVID-19. Vilobelimab (VILO), a C5a-specific monoclonal antibody, improved mortality in severe COVID-19 patients (pts) in a Phase 3 multicenter, randomized, double-blind, placebo (PLC)- controlled study [1]. A pharmacokinetic/pharmacodynamic (PK/PD) analysis was undertaken to assess VILO and C5a as well as antidrug antibodies (ADA) levels in the study. Method(s): Forty-six (46) hospitals on four continents randomized 369 COVID-19 pts (VILO [n = 178], PLC [n = 191]) within 48 h of being mechanically ventilated to receive 6, 800 mg infusions of VILO or PLC at a 1:1 ratio on top of standard of care. Blood samples were taken at screening, Day 8 and at hospital discharge for VILO and C5a and at screening and hospital discharge for ADA. Enzyme-linked immunosorbent assays were used to analyze levels. Result(s): Screening blood samples for VILO and C5a were available for VILO (n = 93) and PLC (n = 99) from sites in Western Europe. On Day 8 after 3 infusions, mean VILO trough concentrations were 21799.3- 302972.1 ng/mL (geometric mean 137881.3 ng/mL) (Fig. 1). At screening, C5a was highly elevated and comparable between groups: VILO median 118.3 ng/mL, mean 130.3 ng/mL, PLC median 104.6 ng/mL, mean 123.2 ng/mL. By Day 8, C5a levels were reduced by 84.6% in the VILO group (median 14.5 ng/mL [mean 16.8 ng/mL], p < 0.001) versus a 19.6% increase in the PLC group (median, 119.2 ng/mL, mean 129.8 ng/ mL). Beyond Day 8, though PD sampling was sparse, C5a levels remained elevated for PLC whereas C5a slowly rose but did not reach screening levels for VILO. Treatment-induced ADA were observed in 1 pt in the VILO group (Day 40 discharge) and 1 pt in the PLC group (Day 25 discharge), both appeared independent of treatment. Conclusion(s): The PK/PD analysis shows that VILO efficiently inhibits C5a in pts with severe COVID-19 resulting in a robust clinical effect on mortality reduction without inducing ADA.

Symptoms and Biomarkers of Long Covid in People Living with and without Hiv

Background: HIV is a risk factor for severe acute COVID-19, but it is unknown whether HIV is a risk factor for long COVID. Method(s): We conducted a prospective observational cohort study of US adults with HIV (PWH) and HIV-seronegative adults with first SARS-CoV-2 infection within 4 weeks together with people who never had COVID-19. At enrollment, participants recalled the presence and severity of 49 long COVID-associated symptoms in the month prior to COVID-19. The same symptom survey was administered at 1, 2, 4, and 6 months post-COVID or post-enrollment for never- COVID participants. Post-COVID participants donated blood 1 and 4 months post-COVID, and never-COVID participants donated blood 0-1 times. Antibody titers to 18 coronavirus antigens and levels of 30 cytokines and hormones were quantified (Meso Scale Discovery). The Mann Whitney U test was used to compare continuous variables between groups, and Pearson's chi-squared test for categorical variables. Spearman correlation analyses were used to build networks of associations between cytokines and symptoms. Result(s): 341 participants enrolled between June 2021 and September 2022. Of these, 73 were PWH post-COVID, 121 were HIV-seronegative post-COVID, 78 were PWH never-COVID, and 69 were HIV-seronegative never-COVID. Over 85% of participants were vaccinated prior to COVID-19. Most participants with HIV were male sex at birth (83% post-COVID, 59% never-COVID), on ART ( >95%), with median CD4 counts >500. Over 60% of participants reported 1+ new or worsened symptoms 2-6 months post-COVID, with higher percentages in PWH at 2 months post-COVID (p< 0.05). PWH were more likely to report body ache, pain, confusion, memory problems, and thirst and had higher levels of creatine phosphokinase post-COVID than HIV-seronegative people. SARS-CoV-2 and non-SARS human coronavirus antibody titers did not differ between PWH and HIV-seronegative post-COVID participants. Cytokine associations with each other (network density) were significantly enriched at 1 month post-COVID in both PWH and HIV-seronegative people, with significantly less enrichment at 4 months post-COVID and in never- COVID participants. Levels of four analytes (cortisol, C5a, TGF-beta1, and TIM-3) associated with specific symptoms of long COVID. Conclusion(s): PWH may experience more symptoms post-COVID with a slightly different symptom profile than people without HIV. Inflammatory networks were active in PWH and people without HIV at 1 month post-COVID.

Phase 3 RCT of C5a-Specific Vilobelimab in Severe COVID-19 Pneumonia

Background: Blocking the C5a-C5aR axis in COVID-19 patients could improve outcomes by limiting myeloid cell infiltration in damaged organs and preventing excessive lung inflammation and endothelialitis. Aims and Objectives: Vilobelimab (VILO), an anti-C5a mAb that preserves the membrane attack complex (MAC), was tested in a Phase III adaptively designed multicenter, double-blind placebo (P)-controlled study for survival in critically ill COVID-19 patients. Method(s): COVID-19 pneumonia patients (N=369;VILO n=178, P n=191) within 48 hrs of intubation were randomly assigned to receive 6, 800 mg infusions of VILO or P on top of standard of care. Primary outcome was 28-day allcause mortality. Result(s): 28-day all-cause mortality was 31.7% VILO vs 41.6% P (Kaplan-Meier estimates;Cox regression site stratified, HR 0.73;95%CI:0.50-1.06;P=0.094) with a 22.7% relative mortality reduction to Day 60. In predefined primary outcome analysis without site stratification, VILO significantly reduced 28-day mortality (HR 0.67;95%CI:0.48-0.96;P=0.027);needed to treat number, 10 to save 1. VILO significantly reduced 28-day mortality in severe patients with baseline WHO ordinal scale score of 7 (n=237, HR 0.62;95%CI:0.40-0.95;P=0.028) or severe ARDS/PaO2/FiO2<=100 mmHg (n=98, HR 0.55;95%CI:0.30-0.98;P=0.044) or eGFR<60 mL/min/1.73m2 (n=108, HR 0.55;95%CI:0.31-0.96;P=0.036). Treatment emergent AEs were 90.9% VILO vs 91.0% P. Infections were comparable;VILO (62.9%), P (59.3%). Serious AEs were 58.9% VILO, 63.5% P. Conclusion(s): VILO reduced mortality at 28 to 60 days in severe COVID-19 pneumonia patients with no increase in infections suggesting the importance of targeting C5a while preserving MAC.

The efficacy and safety of complement C5a inhibitors for patients with severe COVID-19: a systematic review and meta-analysis.

BACKGROUND: The clinical efficacy and safety of complement C5a inhibitors for patients with severe COVID-19 remains unclear. METHODS: The PubMed, Embase, Cochrane Library, and ClinicalTrials.gov databases were searched from their inception to 27 September 2022. Only studies that assessed the usefulness of C5a inhibitors for the treatment of patients with severe COVID-19 patients were included. The primary outcome was the risk of 28-day mortality. RESULTS: Six studies, including four randomized controlled trials (RCTs) and two non-RCTs, were included. The study group receiving C5a inhibitors had a significantly lower risk of mortality compared with the control group (23.6% [70/297] vs 39.2% [136/347]; odds ratio [OR], 0.53; 95% confidence interval [CI]: 0.37-0.76; P< 0.001), and no heterogeneity was detected (I2 = 0%; P= 0.58). Compared with control group, the study group was associated with a similar risk of serious adverse events (AEs) (OR, 0.84; 95% CI: 0.57-1.23; P0 = 0.37), infection (OR, 1.46; 95% CI: 0.77-2.79; P= 0.25) and acute kidney injury (OR, 0.89; 95% CI: 0.54-1.46; P= 0.64). CONCLUSION: C5a inhibitors could help reduce the risk of mortality in patients with severe COVID-19 infection while being as safe as placebos. These findings support the promising role of C5a inhibitors in the treatment of severe COVID-19.

Complete response to eculizumab in atypical hemolytic uremic syndrome with homozygous C3 mutation

Case Report: Atypical Hemolytic Uremic Syndrome (atypical HUS) is a rare and severe form of thrombotic microangiopathy (TMA) characterized by thrombocytopenia, intravascular hemolysis, and acute kidney injury with an incidence of 1 per million.1 Dysregulation and overactivation of the complement alternative pathway due to genetic mutations have been detected in 40-60% of patients with sporadic or familial atypical HUS.2,4 Triggers include viral illness, pregnancy, malignancy, sepsis, or sporadically with no known inciting event.1 Atypical HUS is a severe disease with a 2-10% risk of mortality, 33% risk of end-stage renal failure, and 50% chance of relapse.5 A 24-year-old female with prior history of atypical HUS at the age of 16 (with response to plasmapheresis) presented to the ER with a 5-day history of fever, chills, sore throat, nausea, vomiting, and dark urine. She tested positive for COVID-19. The exam revealed scleral icterus and scattered petechiae. Labs demonstrated nadir hemoglobin (Hgb) of 9.2 g/dL, platelet count of 52 000k/uL, haptoglobin < 30 mg/dL, peak LDH 1128U/L and creatinine 4.62 mg/dL. Urinalysis is consistent with hemoglobinuria. Schistocytes were noted on the peripheral smear. Rapid streptococcal antigen test and C3, C4, and IgA levels were unremarkable. Chest X-Ray, X-ray KUB, and ultrasound abdomen were unremarkable. The pregnancy test was negative. ADAMTS13 was >100%. Genetic analysis after the initial episode at age 16 revealed autosomal recessive inheritance c.193A > c gene mutations in C3. The patient received IV fluids, ceftriaxone for cystitis, and two units of Fresh Frozen Plasma. She initiated treatment with eculizumab. She also received the MENVEO and meningitis B vaccine per protocol due to the risk of meningitis from terminal complement deficiencies. After 4 infusions of eculizumab, patient's labs improved to platelet count of 307 000 k/uL, Hgb 12.2 g/ dL (nadir 9.2 g/dL), haptoglobin 78 mg/dL normalization of LDH and improved creatinine. Atypical HUS is a rare form of TMAwith mutations in C3 noted in 5% of cases. Complement cascade dysfunction leads to endothelial deposits and microvasculature damage. The resulting prothrombotic state causes obstructive microvascular thrombi predominantly affecting the kidneys but can cause multiorgan dysfunction. The SARS-CoV-2 virus may precipitate atypical HUS relapse due to endothelial damage and complement activation further intensified in patients with existing complement aberrations. Plasma exchange remains a standard of care for atypical HUS, as it effectively removes the antibodies and other proteins. Eculizumab a humanized monoclonal IgG antibody binds to complement proteins, preventing cleavage into C5a and C5b blocking C5b-9(MAC) activation. In patients with CFH, CFI, C3, and CFB mutations, eculizumab is the preferred intervention. Copyright © 2023 Southern Society for Clinical Investigation.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Anti-C5a Antibody BDB-001 for Severe COVID-19: A Randomized, Double-Blind, Placebo-Controlled Phase 1 Clinical Trial in Healthy Chinese Adults.

INTRODUCTION: Severe Coronavirus Disease 2019 (COVID-19) progresses with inflammation and coagulation, due to an overactive complement system. Complement component 5a (C5a) plays a key role in the complement system to trigger a powerful "cytokine and chemokine storm" in viral infection. BDB-001, a recombinant human immunoglobulin G4 (IgG4) that specially binds to C5a, has the potential to inhibit the C5a-triggered cytokine storm in treating COVID-19 patients and other inflammation diseases. Here, we have explored its safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adults. This trial is registered with http://www.chinadrugtrials.org.cn/(CTR20200429 ). METHODS: Thirty-two enrolled participants were randomized into three single-dose cohorts (2, 4, and 8 mg/kg) and 1 multi-dose cohort (4 mg/kg), and received either BDB-001 or placebo (3:1) double-blindly. The safety and tolerability after administration were evaluated for 21 days for single-dose cohorts and 28 days for the multi-dose cohort. The pharmacokinetics of BDB-001 in plasma and pharmacodynamics as free C5a in plasma were analyzed. RESULTS: The incidence of drug-related adverse events (AEs) was low, and all AEs were mild or moderate: neither AEs ≥ 3 (NCI-Common Terminology Criteria For Adverse Events, CTCAE 5.0) nor serious adverse events (SAEs) were found. The area under the concentration-time curve from time zero to 480 h (AUC0-480h), that from time zero to infinity (AUCinf), and peak plasma concentration ©max) increased dose-dependently from 2 to 8 mg/kg in the single-dose cohorts and were characterized by a nonlinear pharmacokinetics of target-mediated drug disposal (TMDD). The accumulation index by AUC0-tau after five administrations (4 mg/kg) from the multi-dose cohort was 6.42, suggesting an accumulation effect. Furthermore, inhibition of C5a at the plasma level was observed. CONCLUSION: The results of this phase I study supported that BDB-001 is a potent anti-C5a inhibitor with safety, tolerability, and no immunogenicity. TRIAL REGISTRATION NUMBER: CTR20200429.

Anti-C5a antibody vilobelimab treatment and the effect on biomarkers of inflammation and coagulation in patients with severe COVID-19: a substudy of the phase 2 PANAMO trial.

We recently reported in the phase 3 PANAMO trial that selectively blocking complement 5a (C5a) with vilobelimab led to improved survival in critically ill COVID-19 patients. C5a is an important contributor to the innate immune system and can also activate the coagulation system. High C5a levels have been reported in severely ill COVID-19 patients and correlate with disease severity and mortality. Previously, we assessed the potential benefit and safety of vilobelimab in severe COVID-19 patients. In the current substudy of the phase 2 PANAMO trial, we aim to explore the effects of vilobelimab on various biomarkers of inflammation and coagulation. Between March 31 and April 24, 2020, 17 patients with severe COVID-19 pneumonia were enrolled in an exploratory, open-label, randomised phase 2 trial. Blood markers of complement, endothelial activation, epithelial barrier disruption, inflammation, neutrophil activation, neutrophil extracellular trap (NET) formation and coagulopathy were measured using enzyme-linked immunosorbent assay (ELISA) or utilizing the Luminex platform. During the first 15 days after inclusion, change in biomarker concentrations between the two groups were modelled with linear mixed-effects models with spatial splines and compared. Eight patients were randomized to vilobelimab treatment plus best supportive care (BSC) and nine patients were randomized to BSC only. A significant decrease over time was seen in the vilobelimab plus BSC group for C5a compared to the BSC only group (p < 0.001). ADAMTS13 levels decreased over time in the BSC only group compared to the vilobelimab plus BSC group (p < 0.01) and interleukin-8 (IL-8) levels were statistically more suppressed in the vilobelimab plus BSC group compared to the BSC group (p = 0.03). Our preliminary results show that C5a inhibition decreases the inflammatory response and hypercoagulability, which likely explains the beneficial effect of vilobelimab in severe COVID-19 patients. Validation of these results in a larger sample size is warranted.

X-ray diffraction data for the C5a-peptidase mutant with modified activity and specificity.

The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Engineering of ScpA to enhance its potential as a therapeutic will require detailed examination of the basis for its highly selective activity. The emerging view of ScpA and related subtilases is that selection of their substrates is a dynamic two-step process involving flexibility in the domains around the active site and in the C-ter of the substrate. Surface plasmon resonance (SPR) analyses of the ScpA-hC5a interaction have shown that high affinity binding of the substrate is driven by electrostatic interactions between an exosite on the Fn2 domain of the enzyme and the bulky N-ter cleavage product (PN, 'core' residues 1-67) of C5a [1]. Introduction of a D783A mutation in the Fn2 exosite, located approximately 50 Å from the catalytic serine, was shown to significantly reduce substrate binding affinity and k cat of the enzyme. X-ray crystallographic studies on the D783A mutant (ScpAD783A) were undertaken to better interpret the impact of this mutation on the specificity and activity of ScpA. Here we present the 1.9 Å X-ray diffraction data for ScpAD783A and the molecular replacement solution for the structure. Both raw diffraction images and coordinates have been made available on public databases. Additional details on the related SPR and enzyme kinetics analyses on ScpAD783A reported in Jain et al. [2].

Tissue factor in COVID-19-associated coagulopathy.

Evidence of micro- and macro-thrombi in the arteries and veins of critically ill COVID-19 patients and in autopsies highlight the occurrence of COVID-19-associated coagulopathy (CAC). Clinical findings of critically ill COVID-19 patients point to various mechanisms for CAC; however, the definitive underlying cause is unclear. Multiple factors may contribute to the prothrombotic state in patients with COVID-19. Aberrant expression of tissue factor (TF), an initiator of the extrinsic coagulation pathway, leads to thrombotic complications during injury, inflammation, and infections. Clinical evidence suggests that TF-dependent coagulation activation likely plays a role in CAC. Multiple factors could trigger abnormal TF expression and coagulation activation in patients with severe COVID-19 infection. Proinflammatory cytokines that are highly elevated in COVID-19 (IL-1ß, IL-6 and TNF-α) are known induce TF expression on leukocytes (e.g. monocytes, macrophages) and non-immune cells (e.g. endothelium, epithelium) in other conditions. Antiphospholipid antibodies, TF-positive extracellular vesicles, pattern recognition receptor (PRR) pathways and complement activation are all candidate factors that could trigger TF-dependent procoagulant activity. In addition, coagulation factors, such as thrombin, may further potentiate the induction of TF via protease-activated receptors on cells. In this systematic review, with other viral infections, we discuss potential mechanisms and cell-type-specific expressions of TF during SARS-CoV-2 infection and its role in the development of CAC.

C5a elevation in convalescents from severe COVID-19 is not associated with early complement activation markers C3bBbP or C4d.

Numerous publications have underlined the link between complement C5a and the clinical course of COVID-19. We previously reported that levels of C5a remain high in the group of severely ill patients up to 90 days after hospital discharge. We have now evaluated which complement pathway fuels the elevated levels of C5a during hospitalization and follow-up. The alternative pathway (AP) activation marker C3bBbP and the soluble fraction of C4d, a footprint of the classical/lectin (CP/LP) pathway, were assessed by immunoenzymatic assay in a total of 188 serial samples from 49 patients infected with SARS-CoV-2. Unlike C5a, neither C3bBbP nor C4d readouts rose proportionally to the severity of the disease. Detailed correlation analyses in hospitalization and follow-up samples collected from patients of different disease severity showed significant positive correlations of AP and CP/LP markers with C5a in certain groups, except for the follow-up samples of the patients who suffered from highly severe COVID-19 and presented the highest C5a readouts. In conclusion, there is not a clear link between persistently high levels of C5a after hospital discharge and markers of upstream complement activation, suggesting the existence of a non-canonical source of C5a in patients with a severe course of COVID-19.

ASTHMA, AIRWAY INFLAMMATION AND PTSD ASSOCIATION: RELEVANT MODELS AND A ROLE OF IL17A

Background: There is a growing interest in airway inflammation and mental health. Recent genetic and epidemiological evidence supports an association between PTSD and asthma however, contributory immune mediators/mechanisms are unclear. Recent work from our group employs mouse aeroallergen, house dust mite (HDM) models to examine the role of severe asthma linked inflammatory T helper cells, Th17 and interleukin 17 (IL-17A) in regulating PTSD-relevant behaviors. Methods: A combination of behavioral, immunological, transgenic and transcriptomic approaches were used. 1) BALBc-C5a receptor treatment that shifts Th2 mild asthma phenotype to Th17/IL17a expansion and robust airway inflammation;2) IL-17a receptor knockout mice and 3) RNAseq transcriptomics of cortical and blood brain barrier compromised area, subfornical organ (SFO) tissue was performed. Fear conditioning and extinction was assessed as a PTSD-relevant behavior. Results: Induction of Th17/IL-17 in the BALBc/anti-C5aR1 treated mice resulted in compromised fear extinction and increased fear reinstatement. Absence of IL-17 signaling in IL17Ra deficient mice attenuated HDM effects on fear extinction. Preliminary evidence suggests a potential of the SFO in translating HDM effects to the medial prefrontal cortex, an area regulating fear extinction. Transcriptomic analyses revealed modulation of immune T cell-targeted signaling pathways within the SFO in mice with Th17A expansion. Conclusion: Overall, our work provides novel insights on mechanisms by which mediators of severe airway inflammation, Th17/IL17A regulate fear memory of relevance to PTSD. Beyond asthma-PTSD, our findings have relevant implications for other pulmonary (e.g. COVID-19) and autoimmune inflammatory conditions and mental health.

Exosite binding modulates the specificity of the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease.

The C5a peptidase from Streptococcus pyogenes (ScpA) is a highly specific enzyme with potential therapeutic value. ScpA is a good model for studying determinants of specificity in the multidomain immunomodulatory enzymes (IMEs), which comprise a large family of bacterial surface proteases. The surface exposed region of ScpA has 5 main domains which includes 3 C-terminal Fn3-like domains (Fn1, Fn2 and Fn3) (Kagawa et al. 2009). Progressive deletion of the Fn3-like domains from the C-ter resulted in loss of enzyme activity and showed an important role for the Fn2 domain in enzyme function. Functional investigation of specific acidic residues on the Fn2 domain identified 3 residues 30-50 Å from the catalytic site (D783, E864 and D889) which impacted to differing degrees on binding and on catalysis, supporting the presence of an exosite on the Fn2. In particular, residue D783 was observed to impact on both substrate binding affinity and the activity of ScpA. A double mutant cycle analysis showed energetic coupling between the targeted ScpA residues and residues in the core portion (residues 1-67) of the C5a substrate. The data supports the presence of a communication network between the active site and the exosite on Fn2. These findings provide a basis for rational engineering of this important enzyme family to enhance stability, activity and/or specificity.

[Biomarkers and functional tests in immediate hypersensitivity to SARS-CoV-2 mRNA vaccines]. / Biomarqueurs et tests fonctionnels dans l'hypersensibilité immédiate aux vaccins ARNm dirigés contre le SARS-CoV-2.

Hypersensitivity to mRNA vaccines directed against SARS-CoV-2 are rare. They may be related to an IgE-dependent mechanism involving PEG contained in vaccines in the form of liposomes. Direct activation of the classical complement pathway (CARPA) has also been strongly suspected. In addition to skin tests, biomarkers have been proposed: anti-PEG antibodies, determination of anaphylatoxins C5a and C3a, or soluble complex C5b-9. Anti-PEG antibodies can be measured by non-standardized in house methods; their presence in post-vaccination reactions against SARS-CoV2 has not been confirmed by all the studies as well as for complement proteins. Mast cell mediators (histamine and tryptase) could rarely be assayed at the time of reaction and their increase is inconstant depending on studies. A slightly elevated baseline tryptase level in some patients suggests that hyper-alphatryptasemia might be involved. A basophil activation test (BAT) can be performed but the results are still preliminary depending on the allergens used: PEG, PEG in the form of liposomes or vaccine itself. The lack of positivity of skin tests in some patients even though basophils are able to be activated in the presence of the same allergen confirms the hypothesis in this case of a possible non-IgE-dependent phenomenon. In conclusion, concerning the exploration of immediate reactions to mRNA vaccines directed against SARS-CoV-2, the place of biological markers requires additional studies in order to better identify the actors and mechanisms involved.

Biomarker Levels Concur with a Risk Stratification Criteria in Covid-19 Pneumonia, a Role for a Prognostic-Tool for Identification of Clinical Deteriorators

Rationale: COVID-19 patients present with a number of clinical symptoms ranging from mild, moderate to severe, while only a subgroup of patients, who requires high-dependency critical care resources, accounts for most of the COVID-19 associated health care expenditure and death. A reliable prognostic tool is therefore required to identify patients at risk of developing severe COVID-19 pneumonia. To address this unmet need, we tested a wide range of potentially important peripheral blood biomarkers in a group of clinically risk-stratified COVID-19 patients in order to identify most relevant candidate biomarker(s) predictive of disease progression. Methods: Patients and healthy controls recruited to this study are summarised in Figure 1. Biomarkers levels were analysed using ANOVA across the severity groups. Spearman-correlation coefficients against pairs of average levels from each biomarker within severity-group and healthy controls were assembled into a 76x76 matrix and agglomerative hierarchical clustering was applied to generate the final heatmaps. Linear-discriminant analysis (LDA) was carried out on a reduced optimised set of biomarkers to explore the boundaries between the clinical severity groups.Results: Degree of lymphopaenia, neutrophil levels, TNF-α, INR-levels, and pro-inflammatory cytokines;IL6, IL8, CXCL9 and D-dimers were significantly increased in COVD-19 patients compared to healthy controls (p<0.05, 95% C.I.). C3a and C5 was significantly elevated in all categories of severity compared to healthy controls (p<0.05), C5a levels were significantly different between “moderate” and “severe” categories (p<0.01). sC5b-9 was significantly elevated in the “moderate” and “severe” category of patients compared to healthy controls (p<0.001).Heatmap analysis demonstrated distinct visual differences of biomarker profiles between the clinical severity groups. LDA on the deteriorators, non-deteriorators and healthy volunteers as a combined function of the predictor variables: C3, eosinophil-counts, granulocyte colony-stimulating factor (G-CSF), fractalkine, IL10, IL27, LTB4, lymphocyte count, MIG/CXCL9, M-CSF, platelet count and sC5b-9 showed clear separation between the groups based on biomarker/blood-count levels.Conclusions: Diagnostic and clinical assessments followed by robust statistical and machine learning approaches could identify peripheral blood biomarkers for prognostic stratification of patients in COVID-19. Our results would be helpful for clinicians and supports the use of point of care devices that can quantify multiple analytes. (Lui G, et al., Pointof- care detection of cytokines in cytokine storm management and beyond: Significance and challenges. VIEW. 2021;2: 1-20.). Such would allow for more efficient management and resource allocation. 1 (Figure Presented).

Compassionate Use of an Anti-C5 Inhibitor, Nomacopan, Positively Influenced the Course and Progression of Disease in Covid-19

Background Originally derived from tick-saliva, nomacopan is a first-in-class dual inhibitor of leukotriene B4 (LTB4) and complement C5. Nomacopan (Coversin) is currently in Phase III development for bullous pemphigoid and HSCT-TMA. In this study, we used nomacopan to treat a small cohort of COVID-19 patients on a compassionate basis. We concurrently present data from a study of biomarkers within a larger cohort of COVID-19 patients, where hyperinflammatory pathways due to complement are highlighted. Methods Patients, healthy-controls and sub-groups recruited to this study are summarised in Figure 1. Betweengroup comparisons in demographic, clinical and biomarker levels were carried out using Kruskal-Wallis and rank-Wilcoxon tests. ROX. SpO2Seven patients (six males and one female) in the CORONET study were treated with nomacopan (1st initial subcutaneous-dose: 45mg of nomacopan (t1/2 = 2.5 hrs), + 2 doses;45 mg, 12-hourly. Subsequently, patients were administered 45mg, od for 12 days. Antibiotic prophylaxis was co-administered. Results ROX indices for patients at enrolment within the CASCADE and CORONET studies were lower than that for normal-healthy individuals, with SpO2 <93%, admitted to ICU or COVID-19 Unit with suspected COVID-19 pneumonia and not on invasive mechanical ventilation on recruitment. Average values for SOFA and NEWS scores were significantly different (p<0.05) between the clinical severities. Values for SOFA and NEWS score were not available for the CORONET study patients.CH50, sC5b-9, C5, C5a, C3, and C3a levels were elevated significantly in CASCADE patients (p<0.05, C.I. 95%).Of the seven patients in the CORONET study, six survived, one (female) died, due to unforeseen circumstances (three days delay to get treatment delivered) from start of symptom onset before starting nomacopan treatment. Conclusion The result of this combined study shows that COVID-19 patients, admitted to hospital with significant symptoms of respiratory difficulty, demonstrated increased circulating levels of components of the complement cascade, potentially linked to lung damage leading to fatality. Interestingly, C5-levels (target of nomacopan) was increased, validating the rationale for anti-C5 treatment of COVID-19 patients. Nomacopan treatment was associated with no noticeable adverse event and without highly elevated as associated with normal C5 and C5a levels. (Figure Presented).

2nd Toronto Complement Conference

The proceedings contain 17 papers. The topics discussed include: a helix-swapped C3d dimer mediated by immune evasion protein Sbi hints at a novel s. aureus complement modulation strategy;the role of factor H in macrophages;an antibody targeting complement factor H causes anti-tumor immunity through B-cell activation;C5aR2 deficiency ameliorates inflammation in antibody transfer experimental epidermolysis Bullosa Acquisita and suggests regulating action on the decisive C5a receptor 1;clinical and biomarker characteristics of patients with C3G enrolled in two phase 2 studies investigating the factor D inhibitor Danicopan;perceiver-based machine learning diagnosis of TMA in renal biopsies;and recurrence of atypical hemolytic uremic syndrome After COVID-19 vaccination.

Asthma and PTSD association: relevant models and a role of IL17AA

Background: Strong genetic and epidemiological evidence supports an asthma-PTSD association, however, contributory immune mediators/mechanisms are unclear. Here, we examined a direct role of severe asthma associated Th17/ IL-17A in regulating PTSD-relevant behaviors using mouse aeroallergen house dust mite (HDM) models. Methods: 3 strategies were used: 1) BALBc-C5a receptor treatment that shifts Th2 mild asthma phenotype to Th17/IL17a expansion;2) IL-17a receptor knockout mice and 3) sufficiency testing with intracerebroventricular (ICV) administration of recombinant IL17a effects on behavior. Fear conditioning and extinction, maze exploration and social behaviors were assessed. Results: Absence of IL-17 signaling attenuated HDM effects on fear extinction while induction of Th17/IL-17 in the BALBc/anti-C5aR1 treated mice resulted in compromised fear extinction. ICV IL-17a promoted an anxiogenic phenotype and impaired social behaviors. Preliminary evidence suggests a role of cortical mechanisms (under investigation). Conclusion: Overall, our work highlights severe asthma inflammatory mediator IL17a in regulating PTSD-relevant behaviors. Beyond asthma-PTSD, our findings have relevant implications for other pulmonary (e.g. COVID-19) and autoimmune inflammatory conditions and PTSD risk.

Complement C5a and Clinical Markers as Predictors of COVID-19 Disease Severity and Mortality in a Multi-Ethnic Population.

Coronavirus disease-2019 (COVID-19) was declared as a pandemic by WHO in March 2020. SARS-CoV-2 causes a wide range of illness from asymptomatic to life-threatening. There is an essential need to identify biomarkers to predict disease severity and mortality during the earlier stages of the disease, aiding treatment and allocation of resources to improve survival. The aim of this study was to identify at the time of SARS-COV-2 infection patients at high risk of developing severe disease associated with low survival using blood parameters, including inflammation and coagulation mediators, vital signs, and pre-existing comorbidities. This cohort included 89 multi-ethnic COVID-19 patients recruited between July 14th and October 20th 2020 in Doha, Qatar. According to clinical severity, patients were grouped into severe (n=33), mild (n=33) and asymptomatic (n=23). Common routine tests such as complete blood count (CBC), glucose, electrolytes, liver and kidney function parameters and markers of inflammation, thrombosis and endothelial dysfunction including complement component split product C5a, Interleukin-6, ferritin and C-reactive protein were measured at the time COVID-19 infection was confirmed. Correlation tests suggest that C5a is a predictive marker of disease severity and mortality, in addition to 40 biological and physiological parameters that were found statistically significant between survivors and non-survivors. Survival analysis showed that high C5a levels, hypoalbuminemia, lymphopenia, elevated procalcitonin, neutrophilic leukocytosis, acute anemia along with increased acute kidney and hepatocellular injury markers were associated with a higher risk of death in COVID-19 patients. Altogether, we created a prognostic classification model, the CAL model (C5a, Albumin, and Lymphocyte count) to predict severity with significant accuracy. Stratification of patients using the CAL model could help in the identification of patients likely to develop severe symptoms in advance so that treatments can be targeted accordingly.