A New Registry for Genetic Diagnosis of Inborn Errors of Immunity within the Military Health System

Genotypic definition of monogenic inborn errors of immunity (IEIs) continues to accelerate with broader access to next generation sequencing, underscoring this aggregated group of disorders as a major health burden impacting both civilian and military populations. At an estimated prevalence of 1 in 1200 individuals, IEIs affect ~8,000 patients within the Military Health System (MHS). Despite access to targeted gene/exome panels at military treatment facilities, most affected patients never receive a definitive genetic diagnosis that would significantly improve clinical care. To address this gap, we established the first registry of IEI patients within the MHS with the goal of identifying known and novel pathogenic genetic defects to increase diagnosis rates and enhance clinical care. Using the registry, a research protocol was opened in July 2022. Since July we have enrolled 75 IEI patients encompassing a breadth of phenotypes including severe and recurrent infections, bone marrow failure, autoimmunity/autoinflammation, atopic disease, and malignancy. Enrolled patients provide blood and bone marrow samples for whole genome, ultra-deep targeted panel and comprehensive transcriptome sequencing, plus cryopreservation of peripheral blood mononuclear cells for future functional studies. We are also implementing and developing analytical methods for identifying and interrogating non-coding and structural variants. Suspected pathogenic variants are adjudicated by a clinical molecular geneticist using state-of-the-art analysis pipelines. These analyses subsequently inform in vitro experiments to validate causative mutations using cell reporter systems and primary patient cells. Clinical variant validation and return of genetic results are planned with genetic counseling provided. As a proof of principle, this integrated genetic evaluation pipeline revealed a novel, candidate TLR7 nonsense variant in two adolescent brothers who both endured critical COVID-19 pneumonia, requiring mechanical ventilation and extracorporeal membrane oxygenation. Our protocol is therefore poised to greatly enrich clinical genetics resources available in the MHS for IEI patients, contributing to better diagnosis rates, informed family counseling, and targeted treatments that collectively improve the health and readiness of the military community. Moreover, our efforts should yield new mechanistic insights on immune pathogenesis for a broad variety of known and novel IEIs.Copyright © 2023 Elsevier Inc.

Whole genome sequencing of a German cohort to understand host genetics in COVID-19

Background/Objectives: The disease course upon SARS-CoV-2 infection is highly variable and comprises a range from asymptomatic infection to severe (and even lethal) COVID-19. Genetic factors substantially contribute to this variability, as evidenced by epidemiological studies and recent results from genome-wide association studies (GWAS) as well as sequencing-based approaches. The host genetics group of the German COVID-19 OMICs Initiative (DeCOI) has been founded with the aim to identify additional genetic variants that influence COVID-19 severity through whole genome sequencing (WGS) analyses. Method(s): Until January 2022, WGS has been performed on approximately 1200 individuals affected by COVID-19. Result(s): The most recent data freeze comprised 952 individuals. In this dataset, no carrier of a deleterious protein-altering variant has been detected in TLR7, which is the only conclusive risk gene for severe COVID-19. Applying a gene-based association test of rare variants to the subcohort of European individuals (n = 752, mean age: 56 years, females: 44%), including 199 severely affected individuals, we did not observe any significant association after correction for multiple testing. Exome-wide association analysis of common variants in this subcohort replicated the GWAS-locus on chromosome 3. Conclusion(s): With this ongoing work, we are contributing to international efforts to elucidate the host genetics of COVID-19, also by sharing our summary statistics for meta-analyses. Currently, we are sequencing additional severely affected individuals and we are refining analytical strategies, which will also include the joint analysis of common and rare variants at genomewide scale.

"Actionable" host genetic markers for repurposing "add-on therapy" in COVID-19

Background/Objectives: COVID-19 still represents a lifethreatening disease in individuals with a specific genetic background. We successfully applied a new Machine Learning method on WES data to extract a set of coding variants relevant for COVID- 19 severity. We aim to identify personalized add-on therapy. Method(s): A subset of identified variants, "actionable" by repurposed drugs, were functionally tested by in vitro and in vivo experiments. Result(s): Males with either rare loss of function variants in the TLR7 gene or L412F polymorphism in the TLR3 gene benefit from IFN-gamma, which is specifically defective in activated PBMCs, restoring innate immunity. Females heterozygous for rare variants in the ADAMTS13 gene and males with D603N homozygous polymorphism in the SELP gene benefit from Caplacizumab, which reduces vWF aggregation and thrombus formation. Males with either the low-frequency gain of function variant T201M in CYP19A1 gene or with poly-Q repeats >=23 in the AR gene benefit from Letrozole, an aromatase inhibitor, which restores normal testosterone levels, reducing inflammation and which rescues male golden hamsters from severe COVID-19. Conclusion(s): By adding these commonly used drugs to standard of care of selected patients, the rate of intubation is expected to decrease consistently, especially in patients with high penetrance rare genetic markers, mitigating the effect of the pandemic with a significant impact on the healthcare system.

Role of toll-like receptors in the pathogenesis of COVID-19: Current and future perspectives

The coronavirus disease 2019 (COVID-19) pandemic underlines a persistent threat of respiratory tract infectious diseases and warrants preparedness for a rapid response. At present, COVID-19 has had a serious social impact and imposed a heavy global burden on public health. The exact pathogenesis of COVID-19 has not been fully elucidated. Since the outbreak of COVID-19, a renewed attention has been brought to Toll-like receptors (TLRs). Available data and new findings have demonstrated that the interaction of human TLRs and SARS-CoV-2 is a vital mediator of COVID-19 immunopathogenesis. TLRs such as TLR2, 4, 7 and 8 are potentially important in viral combat and activation of immunity in patients with COVID-19. Therapeutics targeting TLRs are currently considered promising options against the pandemic. A number of TLR-targeting immunotherapeutics are now being investigated in preclinical studies and different phases of clinical trials. In addition, innovative vaccines based on TLRs under development could be a promising approach for building a new generation of vaccines to solve the current challenges. In this review, we summarize recent progress in the role of TLRs in COVID-19, focusing the new candidate drugs targeting TLRs, the current technology and potential paths forward for employing TLR agonists as vaccine adjuvants.Copyright © 2023 The Scandinavian Foundation for Immunology.

Association of Toll-Like Receptor 7 and 8 Polymorphisms with Covid-19 Severity

Objectives: Toll-like receptors (TLRs) are an important family of receptors that recognize infectious agents and play an important role in the innate immune system. TLRs are a potential candidate to control infection in the early stages of the disease and to produce vaccines against SARS-CoV-2. In addition, studies have suggested that TLR polymorphisms are also associated with antiviral responses against SARS-CoV-2. Therefore, we aimed to investigate the relationship of TLR7 and TLR8 polymorphisms with COVID-19 disease prognosis. Materials-Methods: A total of 120 COVID-19 patients, including 40 outpatients, 40 patients with mild and moderate clinical status, hospitalized and severe pneumonia, and 40 patients followed in the intensive care unit (ICU), were included in the study. The classification of disease severity was made according to WHO criteria. TLR7 (rs179009), TLR8 -129 C/G (rs3764879) and TLR8 Met1Val (rs3764880) polymorphisms were genotyped using the PCR-RFLP method. Result(s): Since TLR7 and TLR8 are located on the X chromosome, men and women were analyzed separately. There was no significant difference between the groups in terms of 3 polymorphisms in males. On the other hand, in women, individuals carrying AG genotype and G allele for TLR8 Met1Val polymorphism were found at a higher rate in patients hospitalized in ICU than in patients followed in the service (p <0.05). In terms of the other two polymorphisms, no significant difference was found between the groups in women. Conclusion(s): We suggest that the AG genotype and G allele of TLR8 Met1Val polymorphism can be considered as an important risk factor that increases the severity of the disease in women.

Enhanced TLR7 immunity drives innate protection against SARS-CoV-2 with chilblains as collateral damage

Outbreaks of chilblains, a hallmark sign of type I interferonopathies, have been reported during the COVID-19 pandemic. These cases occurred mostly in patients who were asymptomatic and showed negative results from PCR and serology tests for SARS-CoV-2. We hypothesized that chilblain patients are predisposed to mount a robust innate immunity against the virus, which clinically manifests as chilblains and promotes early viral clearance, thereby preventing pulmonary disease and precluding adaptive responses. By profiling skin lesions in the early stage following chilblain onset, we uncover a transient IRF7-dependent type I interferon (IFN) signature that is driven by the acral infiltration of systemically activated plasmacytoid dendritic cells (pDCs). Patients' peripheral blood mononuclear cells (PBMCs) demonstrate increased production of IFNalpha when exposed to SARS-CoV-2 and influenza A, but not herpes simplex virus 1 (HSV-1), indicating a heightened ability to detect RNA -but not DNA- viruses. Further investigations revealed enhanced responsiveness of pDCs in chilblain patients to the RNA sensor TLR7, but not the DNA sensor TLR9. Collectively, our study establishes a two-step model for the immunopathology of SARS-CoV-2-related chilblains: enhanced TLR7 immunity in pDCs, likely triggered by SARS-CoV-2 exposure at the mucosal site, leads to prompt viral clearance, which explains the lack of infection markers in most cases. Subsequently, systemic spread of activated pDCs and infiltration of the toes in response to mechanical stress or acral coldness, may result in IFN-mediated tissue damage with development of chilblains.Copyright © 2023

Polysaturated fatty acids and their derivatives regulate respiratory infections.

The regulation of inflammation is a complex pathophysiological process that depends on the production of oxygenated lipid derivatives essential polyunsaturated fatty acids, like omega-3 and omega-6, among which are the lipoxins resolvins and protectins, called specialized pro-resolving lipid mediators (SPM). Their activity is associated with the control of respiratory infection processes to modulate the production of proinflammatory cytokines, avoiding damage due to inflammation-associated necrosis, reducing microbial loads, and promoting tissue remodeling. Therefore, we review some of the biochemical, physiological and immunological aspects of SPM in the regulation of inflammation in respiratory infections.Copyright © 2022, Instituto Nacional de Enfermedades Respiratorias. All rights reserved.

Frequency of IRF5+ dendritic cells is associated with the TLR7-induced inflammatory cytokine response in SARS-CoV-2 infection.

The SARS-CoV-2 infection leads to enhanced inflammation driven by innate immune responses. Upon TLR7 stimulation, dendritic cells (DC) mediate the production of inflammatory cytokines, and in particular of type I interferons (IFN). Especially in DCs, IRF5 is a key transcription factor that regulates pathogen-induced immune responses via activation of the MyD88-dependent TLR signaling pathway. In the current study, the frequencies of IRF5+ DCs and the association with innate cytokine responses in SARS-CoV-2 infected individuals with different disease courses were investigated. In addition to a decreased number of mDC and pDC subsets, we could show reduced relative IRF5+ frequencies in mDCs of SARS-CoV-2 infected individuals compared with healthy donors. Functionally, mDCs of COVID-19 patients produced lower levels of IL-6 in response to in vitro TLR7 stimulation. IRF5+ mDCs more frequently produced IL-6 and TNF-α compared to their IRF5- counterparts upon TLR7 ligation. The correlation of IRF5+ mDCs with the frequencies of IL-6 and TNF-α producing mDCs were indicators for a role of IRF5 in the regulation of cytokine responses in mDCs. In conclusion, our data provide further insights into the underlying mechanisms of TLR7-dependent immune dysfunction and identify IRF5 as a potential immunomodulatory target in SARS-CoV-2 infection.

AMP5A modulates Toll-like receptors 7 and 8 single-stranded RNA immune responses in PMA-differentiated THP-1 and PBMC.

Background: Dysregulation of antiviral immunity has been implicated in the progression of acute respiratory syndrome coronavirus 2 infection into severe cases of coronavirus disease of 2019 (COVID-19). Imbalances in the inflammatory response drive the overabundant production of pro-inflammatory cytokines and chemokines. The low molecular weight fraction of 5% human serum albumin commercial preparation (AMP5A) is a novel biologic drug currently under clinical investigation for the treatment of osteoarthritis and the hyperinflammatory response associated with COVID-19. This study aims to elucidate AMP5A effects following the activation of immune cells with agonists of Toll-like receptor (TLR) 7 and/or 8, which detect ssRNA viral sequences. Methods: CXCL10 ELISAs were used to evaluate the dynamics of myeloid cells activated with CL075 and CL307, agonists of TLR7/8 and TLR7, respectively. In addition, enrichment analysis of gene sets generated by ELISA arrays was utilized to gain insight into the biologic processes underlying the identified differentially expressed cytokine profiles. Finally, relative potency (REP) was employed to confirm the involvement of mechanisms of action paramount to AMP5A activity. Results: AMP5A inhibits the release of CXCL10 from both CL075- and CL307-activated PMA-differentiated THP-1 and peripheral blood mononuclear cells. Furthermore, AMP5A suppresses a distinct set of pro-inflammatory cytokines (including IL-1ß, IL-6, IL-12, and CXCL10) associated with COVID-19 and pro-inflammatory NF-κB activation. REP experiments using antagonists specific for the immunomodulatory transcription factors, peroxisome proliferator-activated receptor γ, and aryl hydrocarbon receptor, also indicate that these pathways are involved in the ability of AMP5A to inhibit CXCL10 release. Conclusion: Due to the biphasic course of COVID-19, therapeutic approaches that augment antiviral immunity may be more beneficial early in infection, whereas later interventions should focus on inflammation suppression. In this study, we show that AMP5A inhibits TLR 7/8 signaling in myeloid cells, resulting in a decrease in inflammatory mediators associated with hyperinflammation and autoimmunity. Furthermore, data demonstrating that AMP5A activates immunomodulatory transcription factors found to be protective in lung disease is provided. These findings suggest that the modes and mechanisms of action of AMP5A are well suited to treat conditions involving dysregulated TLR 7/8 activation.

Intranasal administration of a VLP-based vaccine induces neutralizing antibodies against SARS-CoV-2 and variants of concern

Background: The highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID-19. Methods: In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVTT -RBD, has been optimized to incorporate a universal T helper cell epitope derived from tetanus-toxin and is self-adjuvanted with TLR7/8 ligands. Results: CuMVTT-RBD vaccine elicited a strong systemic RBD- and spike- IgG and IgA antibodies of high avidity. Local immune response was assessed and our results demonstrate a strong mucosal antibody and plasma cell production in lung tissue. Furthermore, the induced systemic antibodies could efficiently recognize and neutralize different variants of concern (VOCs) of mutated SARS-CoV-2 RBDs. Conclusion: Our data demonstrate that intranasal administration of CuMVTT-RBD induces a protective systemic and local specific antibody response against SARS-CoV-2 and its VOCs.

Nanoparticles: Efficient, safe and affordable platforms for developing vaccines against coronaviruses

Introduction: Vaccines need to be rationally designed to be delivered to the immune system for maximizing induction of dynamic immune responses. Virus-like nanoparticles (VLPs) are ideal platforms for such 3D vaccines. Coronaviruses have recently gained a lot of attention, due to the ongoing pandemic caused by SARS-CoV-2 and previous endemics by MERS-CoV. Methods: We have provided proof of concepts in murine models for effective development of VLP-based vaccines against MERS-CoV and SASR-CoV-2. We have used chemical conjugation or genetic fusion techniques to display receptor-binding domain or motif on our immunologically optimized (CuMVTT -VLPs). These VLPs incorporate a tetanus toxin epitope and ssRNA, TLR7/8 ligands. The vaccines were tested in murine models. Results: The vaccines are stable for more than a year at 4°C and highly scalable. Vaccination using subcutaneous or intranasal routes are feasible with nanoparticles. We demonstrated that these vaccines are highly immunogenic in mice as well as rabbits and can induce high avidity antibodies compared to convalescent human sera. Furthermore, the induced antibodies are cross-reactive with different VoCs (in case of SARS-CoV-2). The longevity of the induced immune response lasted longer than 120 days. Conclusion: Collectively, we show that VLP-based vaccines can efficiently induce high specific anti-RBD and spike antibodies that effectively neutralize different Coronaviruses and their VoCs. As Coronaviruses represent a continuous global threat to human health, it seems rational to further develop these vaccines.

PHASE II TRIAL of ENPATORAN in PATIENTS HOSPITALIZED with COVID-19 PNEUMONIA

Background: Enpatoran is a selective and potent dual toll-like receptor (TLR) 7/8 inhibitor in development for the treatment of cutaneous and systemic lupus erythematosus (CLE/SLE). Enpatoran inhibits TLR7/8 activation in vitro and suppresses disease activity in lupus mouse models.1 Enpatoran was well tolerated and had linear pharmacokinetic (PK) parameters in healthy volunteers.2 As TLR7/8 mediate immune responses to single-stranded RNA viruses, including SARS-CoV-2, it was postulated that enpatoran may prevent hyperinfammation and cytokine storm in COVID-19. Objectives: In response to the COVID-19 pandemic, we conducted an exploratory Phase II trial to assess safety and determine whether enpatoran prevents clinical deterioration in patients (pts) hospitalized with COVID-19 pneumonia. PK and pharmacodynamics (PD) of enpatoran were also evaluated. Methods: ANEMONE was a randomized, double-blind, placebo (PBO)-con-trolled study conducted in Brazil, the Philippines, and the USA (NCT04448756). Pts aged 18-75 years, hospitalized with COVID-19 pneumonia (WHO 9-point scale score =4) but not mechanically ventilated, with SpO2 <94% and PaO2/FiO2 ≥150 (FiO2 maximum 0.4) were eligible. Those with a history of uncontrolled illness, active/unstable cardiovascular disease and SARS-CoV-2 vaccination were excluded. Pts received PBO or enpatoran (50 or 100 mg twice daily [BID]) for 14 days, with monitoring to Day 28 and safety follow-up to Day 60. Primary outcomes were safety and time to recovery (WHO 9-point scale ≤3). Clinical deterioration (time to clinical status >4, WHO 9-point scale) was a secondary outcome. Exploratory endpoints were enpatoran and biomarker concentrations (cytokines, C-reactive protein [CRP], D-dimer and interferon gene signature [IFN-GS] scores) assessed over time. Results: 149 pts received either PBO (n=49), or enpatoran 50 mg (n=54) or 100 mg (n=46) BID;88% completed treatment and 86% received concomitant steroids. Median age was 50 years (77% <60 years old), 66% were male, and 50% had ≥1 comorbidity (40% hypertension, 24% diabetes). Overall, 59% pts reported a treatment-emergent adverse event (TEAE) with three non-treatment-related deaths;11% reported a treatment-related TEAE. The proportion of pts in the enpatoran group reporting serious TEAEs was low (50 mg BID 9%;100 mg BID 2%) vs PBO (18%). Gastrointestinal disorders were most common (PBO 8%;50 mg BID 28%;100 mg BID 9%). The primary outcome of time to recovery with enpatoran vs PBO was not met;medians were 3.4-3.9 days. A positive signal in time to clinical deterioration from Day 1 through Day 28 was observed;hazard ratios [95% CI] for enpatoran vs PBO were 0.39 [0.13, 1.15] (50 mg BID) and 0.30 [0.08, 1.08] (100 mg BID). Mean enpatoran exposure was dose-proportional, and PK properties were within expectations. The median (quartile [Q]1-Q3) interleukin 6 (IL-6), CRP and D-dimer baseline concentration across the groups were 5.7 (4.0-13.5) pg/mL, 30.04 (11.40-98.02) and 0.62 (0.39-1.01) mg/L, respectively. Baseline IFN-GS scores were similar across groups. Conclusion: The ANEMONE trial was the frst to evaluate the safety and efficacy of a TLR7/8 inhibitor in an infectious disease for preventing cytokine storm. Enpa-toran up to 100 mg BID for 14 days was well tolerated by patients acutely ill with COVID-19 pneumonia. Time to recovery was not improved with enpatoran, perhaps due to the younger age of patients who had fewer comorbidities compared to those in similar COVID-19 trials. However, there was less likelihood for clinical deterioration with enpatoran than placebo. This trial provides important safety, tolerability, PK and PD data supporting continued development of enpatoran in SLE and CLE (NCT04647708, NCT05162586).

TREK-1 Potassium Channel Activation Protects Against Influenza-A Induced Lung Injury

Introduction and Rationale: No targeted therapies exist that improve the outcomes of patients with Acute Respiratory Distress Syndrome (ARDS), in part to the multifactorial etiology of this devastating disease. Infectious agents remain the most common initiating insults, and besides SARS-CoV-2, Influenza-A virus (IAV) is responsible for more ARDS cases and deaths than any other agent. In fact, IAV increases the risk of mortality in ARDS patients three-fold, and accounts for almost half of all ARDS deaths. We recently identified TREK-1 potassium channels on epithelial cells as important regulators of alveolar inflammation and barrier function, two hallmarks of ARDS, and found that pharmacological activation of TREK-1 protects against hyperoxia-induced lung injury. However, whether TREK-1 channels convey similar protection in a clinically more relevant IAVinduced lung injury model, remains unknown. Methods: We infected adult C57BL/6 wildtype mice intra-tracheally (i.t.) with IAV (PR8 strain;TCID50 400), followed by once-daily i.t. injections (days 5, 6 and 7 post-IAV) with the novel TREK-1 activating compounds ML335 (60mcg/kg), BL1249 (100mcg/kg), or a vehicle control, to create a clinically-relevant treatment model. To evaluate the role of epithelial cells in this model, we infected primary human alveolar epithelial cells (HAEC) with IAV (0.01 pfu) for 24 hours. Endpoint analysis consistent in quantification of quasi-static lung compliance;BAL fluid total protein, cell counts, and ROS concentrations;cytokine levels in BAL fluid and cell supernatants;and HAEC viability (XTT assay). In addition, we measured alterations in epithelial potassium currents (fluorometric FLIPR assays) and in IAV-induced signaling cascades (real-time PCR) following IAV infection and treatment with our TREK-1 activators. Results: Oncedaily treatment of mice with the TREK-1 activating compounds ML335 or BL1249 following IAV infection improved lung compliance, and BAL fluid total protein levels, cell counts, IL-6, CXCL-10, MIP-1alpha, and TNF-alpha concentrations, but not ROS, CCL-2 or IL-10 levels. In HAEC, TREK-1 activation improved IAV-induced IL-6, CXCL-10, and CCL-2 levels, while MIP-1alpha, TNF-alpha and IL-10 levels remained unchanged. XTT assays confirmed that in our model IAV infection did not cause significant cell death. Interestingly, IAV infection decreased HAEC potassium currents, which could be counteracted by TREK-1 activation and cell hyperpolarization. Finally, TREK-1 activationmediated cell hyperpolarization inhibited TLR4- and TNFSF13-mediated downstream signaling in IAV-infected HAEC, whereas NFkB, RIG1, TLR3, and TLR7 signaling was not affected. Conclusions: TREK-1 potassium channel activation may represent a novel approach to protect against IAV-induced acute lung injury.

COVID-19 mRNA vaccines associated with MOG-Ab disease and transverse myelitis: Two case reports

Objective: To describe, to the best of our knowledge, the first case of myelin oligodendrocyte glycoprotein (MOG) antibody syndrome associated with the Moderna COVID-19 mRNA vaccine, and a case of acute transverse myelitis (ATM) associated with the Pfizer COVID-19 mRNA vaccine. Background: Post-vaccination CNS demyelinating syndromes have been reported with different vaccines, most notably the influenza and human papilloma vaccines. Two cases of new-onset multiple sclerosis (MS) and a case of new-onset neuromyelitis optica (NMO) associated with the Pfizer vaccine have been reported. One case of new-onset relapsing-remitting MS was reported after the Moderna vaccine. Design/Methods: NA Results: A 38-year-old man developed left blurry vision, lower extremity weakness/paresthesia and bowel/bladder dysfunction three days after receiving the Moderna vaccine. He was diagnosed with left optic neuritis and longitudinally extensive transverse myelitis;he tested positive for MOG antibody. A 39-year-old woman presented with progressive lower extremity weakness/numbness seven days after receiving the Pfizer vaccine. She was diagnosed with ATM. Both patients improved with intravenous corticosteroids. Conclusions: The association between CNS demyelinating syndromes and vaccination has been reported for many years. The proposed pathogenesis of CNS demyelinating syndromes after vaccines includes molecular mimicry, epitope spreading, bystander activation, polyclonal activation, effects of adjuvants, and depends on vaccine-related factors like type, dose, and route of administration. The adjuvanticity of COVID-19 vaccines is novel in that it involves Toll-like Receptor (TLR) 7 and 9 agonism, and several immune-mediated disorders have been linked to altered nucleic acid metabolism and processing that have stimulated TLR-7 and TLR-9 experimentally. While the risk of CNS demyelinating events is non-negligible, the incidence is very low. The rate of demyelinating events after the COVID-19 infection is higher. Therefore, we feel that the overall benefits of vaccination outweigh the marginal risk. However, providers should be aware of this potential neurological complication of the COVID-19 mRNA vaccines.

COVID-19 susceptibility, severity, clinical outcome and Toll-like receptor (7) mRNA expression driven by TLR7 gene polymorphism (rs3853839) in middle-aged individuals without previous comorbidities.

Background: Toll-like receptors are implicated in the pathophysiology of the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory disease (MERS), according to several studies. The whole-genome sequencing of SARS-CoV-2 revealed that the TLR7 gene could be implicated in the virus's pathogenesis since the virus includes ssRNA patterns that could bind to TLR7. Aim: The purpose of this study was to look into the function of the TLR7 (rs3853839) C/G polymorphism and the expression of TLR7 mRNA transcript in the development, severity and progression of COVID-19. Subjects and methods: A case-control study included 285 participants who were divided into two groups: 150 middle-aged people with COVID 19 who had no previous co-morbidities and 135 healthy volunteers who served as controls. TaqMan test was used to genotype the TLR7 (rs3853839) C/G polymorphism, and real-time PCR was used to determine the relative expression of its mRNA transcript. The level of IL-6 in serum was determined using the ELISA method as an indicator of cytokine storm and COVID-19 severity. Results: The GG genotype was shown to be much more common in COVID-19 patients (38.7%) than controls (4.4%), with an OR of 19.86 (95% CI: 7.85; 50.22) and was linked to disease severity and poor clinical outcomes (hospitalization, respiratory failure, cardiac complications, ICU admission and mechanical ventilation).As a result, the G allele was considerably higher in cases (57.0%), while the C allele was significantly higher in controls (p = 0.001). The GG genotype was found to be substantially more common in patients who were severely/critically unwell. TLR7 mRNA expression levels were significantly higher in COVID-19 patients (2.44 ± 0.89) than in controls (1.06 ± 0.46) (p = 0.001). TLR7 mRNA levels were highest in COVID 19 patients with the GG genotype (rs3853839). Patients with the GG genotype had considerably lower WBC counts, but significantly higher serum ferritin, CRP, IL-6 and D dimer levels (P = 0.045, 0.001, 0.023, 0.033, 0.001, respectively). Conclusion: The GG form of the TLR7 SNP (rs3853839) could be a genetic risk factor for COVID-19 infection, severe illness and poor clinical outcome. TLR7 mRNA expression was also elevated in COVID-19 patients who were severely/critically unwell and had a bad outcome, suggesting that they could be used as COVID-19 prognostic biomarkers.

Do reduced numbers of plasmacytoid dendritic cells contribute to the aggressive clinical course of COVID-19 in chronic lymphocytic leukaemia?

Infections with SARS-CoV-2 have been unduly severe in patients with haematological malignancies, in particular in those with chronic lymphocytic leukaemia (CLL). Based on a series of observations, we propose that an underlying mechanism for the aggressive clinical course of COVID-19 in CLL is a paucity of plasmacytoid dendritic cells (pDCs) in these patients. Indeed, pDCs express Toll-like receptor 7 (TLR7), which together with interferon-regulatory factor 7 (IRF7), enables pDCs to produce large amounts of type I interferons, essential for combating COVID-19. Treatment of CLL with Bruton's tyrosine kinase (BTK) inhibitors increased the number of pDCs, likely secondarily to the reduction in the tumour burden.

Model-informed Dose Selection of Dual Toll-like Receptor 7/8 Inhibitor Enpatoran (M5049) for the Treatment of COVID-19 Pneumonia

Background. Enpatoran, formerly known as M5049, is a potential first-in-class small molecule antagonist of toll-like receptors (TLR) 7 and 8, which may prevent viral-associated hyperinflammatory response and progression to 'cytokine storm' in coronavirus disease 2019 (COVID-19) patients. The objective of this study was to leverage existing population pharmacokinetic/pharmacodynamic (popPK/PD) models for enpatoran to inform dose selection for an accelerated Phase II study in COVID-19 patients with pneumonia. Methods. The popPK/PD models were based on plasma PK and PD biomarker (ex vivo-stimulated interleukin [IL]6 and interferon α [IFNα] secretion) data from the enpatoran first-in-human Phase I study in healthy participants (Port A, et al. Lupus Sci Med 2020;7(Suppl. 1): P135). A two-compartment model describing PK used a sigmoidal Emax model with proportional decrease from baseline characterizing the PD response across the investigated single and multiple daily dose range of 1-200 mg (N=72). Concentrations that inhibited 50% and 90% (IC50/IC90) of cytokine secretion were estimated and stochastic simulations were performed to assess target coverage under different dosing regimens. Results. Simulations suggested that, to achieve maximal inhibition of IL-6 over time, enpatoran PK concentrations would be maintained above the IC90 throughout the dosing interval with doses of 100 mg and 50 mg twice daily in 90% and 30% of participants, respectively. In comparison, IFNα inhibition was predicted to be lower, with IC90 coverage in 60% and 8% of participants with twice daily doses of 100 mg and 50 mg enpatoran, respectively. Conclusion. Utilization of existing popPK/PD models allowed for the accelerated development of enpatoran in COVID-19 to address an unprecedented global pandemic. Rational model-informed dose selection was supported by data from a Phase I study in which there were no safety concerns.

An Updated Review on the Role of Single Nucleotide Polymorphisms in COVID-19 Disease Severity: A Global Aspect.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and recently has become a serious global pandemic. Age, gender, and comorbidities are known to be common risk factors for severe COVID-19 but are not enough to fully explain the magnitude of their effect on the risk of severity of the disease. Single nucleotide polymorphisms (SNPs) in several genes have been reported as a genetic factor contributing to COVID-19 severity. This comprehensive review focuses on the association between SNPs in four important genes and COVID-19 severity in a global aspect. We discuss a total of 39 SNPs in this review: five SNPs in the ABO gene, nine SNPs in the angiotensin-converting enzyme 2 (ACE2) gene, 19 SNPs in the transmembrane protease serine 2 (TMPRSS2) gene, and six SNPs in the toll-like receptor 7 (TLR7) gene. These SNPs data could assist in monitoring an individual's risk of severe COVID-19 disease, and therefore personalized management and pharmaceutical treatment could be planned in COVID-19 patients.

Whole exome sequencing of MIS-C patients: Analysis of genes involved in inborn errors of type I IFN immunity, hemophagocytic lymphohistiocytosis (HLH), Kawasaki disease (KD) and TLR7 gene

Introduction: After spring 2020, a series of reports from Europe and USA described clusters of children, presenting life-threatening multisystem inflammatory syndrome in children (MIS-C), associated with antecedent exposure to SARS-CoV-2 (1). In patients with life threatening COVID-19 3.5% were found to have inborn errors in type I IFN signalling pathway (2). A case series of 4 young patients with severe COVID-19 reported rare loss-of-function variants in the TLR7 gene associated with impaired type I IFN responses (3). Clinically, MIS-C shares features with secondary hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), which were also associated with possible infectious trigger and might share a common genetic cause (4). Objectives: We analysed whether MIS-C patients have an underlying presence of genetic variants in exomes associated with inborn errors of type I IFN immunity, HLH, KD and presence of variants in TLR7 gene. Methods: Blood was drawn from 17 MIS-C patients upon submission into the hospital, DNA from peripheral blood was isolated and whole exome sequencing was performed. Variants in the following genes were investigated: type I IFN immunity (TLR3, UNC93B1, TRAF3, TBK1, IRF3/9, IRF7, IFNAR1/2, STAT1/2, IKBKG, TRIF), HLH (AP3B1, CD27, FADD, FAS, FASLG, HPLH1, ITK, LYST, MAGT1, MYO5A, NLRC4, PRF1, RAB27A, RECQL4, SH2D1A, STX11, STXBP2, UNC13D, XIAP, TNFRSF9, CDC42), KD (ITPKC, CD40, FCGR2A, BLK, CASP3, TRX-CAT1-7, PGBD1, LTA, TSBP1, HLA-DQB1/2, HLA-DOB, IGHV1-69) and TLR7 genes. Analysis was focused on rare (GnomAD<0.01) exonic or splicing variants. Results: No common genetic denominators were found in analysed genes. Five rare variants were observed in four patients (4/17). According to ACMG classification variants of uncertain significance (VUS) were found in LYST (2), IKBKG (1), IRF3 (1) and NLRC4 (1) in heterozygous genotype. No clinical evidence was found in ClinVar database for any of the variants, except for one variant in LYST (c.3931A>G:p.M1311V) with uncertain significance for Chédiak-Higashi syndrome and medium prediction scores. Variants in LYST (c.5990C>G:p.A1997G), NLRC4 (c.772T>C:p.C258R) and IRF3 (c.325G>C: p.G109R) have high CADD, Mutation Taster, Polyphen and SIFT prediction scores. And IKBKG (c.325C>G:p.L109V) variant had medium prediction scores. Conclusion: Our findings suggest that MIS-C patients do not share a rare loss-of-function variant in type I IFN immunity genes, TLR7 gene or genes associated either with HLH or KD. Despite numerous clinical, immunological and genetic research of the MIS-C patients, the syndromes pathogenesis and etiologic cause remain elusive.