The Impact of IFNλ4 on the Adaptive Immune Response to SARS-CoV-2 Infection.

Genetic polymorphisms at the IFNL4 loci are known to influence the clinical outcome of several different infectious diseases. Best described is the association between the IFNL4 genotype and hepatitis C virus clearance. However, an influence of the IFNL4 genotype on the adaptive immune system was suggested by several studies but never investigated in humans. In this cross-sectional study, we have genotyped 201 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive participants for 3 IFNL4 polymorphisms (rs368234815, rs12979860, and rs117648444) and stratified them according to the IFNλ4 activity. Based on this stratification, we investigated the association between the IFNL4 genotype and the antibody as well as the CD8+ T cell response in the acute phase of the SARS-CoV-2 infection. We observed no differences in the genotype distribution compared with a Danish reference cohort or the 1,000 Genome Project, and we were not able to link the IFNL4 genotype to changes in either the antibody or CD8+ T cell responses of these patients.

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

Effect of Age on Innate and Adaptive Immunity in Hospitalized COVID-19 Patients.

An effective but balanced cellular and inflammatory immune response may limit the severity of coronavirus disease (COVID-19), whereas uncontrolled inflammation leads to disease progression. Older age is associated with higher risk of COVID-19 and a worse outcome, but the underlying immunological mechanisms for this age-related difference are not clear. We investigated the impact of age on viral replication, inflammation, and innate and adaptive cellular immune responses in 205 hospitalized COVID-19 patients. During the early symptomatic phase of COVID-19, we found that patients above 65 years had significantly higher viral load, higher levels of proinflammatory markers, and inadequate mobilization and activation of monocytes, dendritic cells, natural killer cells, and CD8 T cells compared to those below 65 years. Our study points toward age-related deficiencies in the innate immune cellular response to SARS-CoV-2 as a potential cause of poorly controlled viral replication and inflammation during the early symptom phase and subsequent disease progression.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Efficacy of the TMPRSS2 inhibitor camostat mesilate in patients hospitalized with Covid-19-a double-blind randomized controlled trial.

BACKGROUND: The trans-membrane protease serine 2 (TMPRSS2) is essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry and infection. Efficacy and safety of TMPRSS2 inhibitors in patients with coronavirus disease 2019 (Covid-19) have not been evaluated in randomized trials. METHODS: We conducted an investigator-initiated, double-blind, randomized, placebo-controlled multicenter trial in patients hospitalized with confirmed SARS-CoV-2 infection from April 4, to December 31, 2020. Within 48 h of admission, participants were randomly assigned in a 2:1 ratio to receive the TMPRSS2 inhibitor camostat mesilate 200 mg three times daily for 5 days or placebo. The primary outcome was time to discharge or clinical improvement measured as ≥2 points improvement on a 7-point ordinal scale. Other outcomes included 30-day mortality, safety and change in oropharyngeal viral load. FINDINGS: 137 patients were assigned to receive camostat mesilate and 68 to placebo. Median time to clinical improvement was 5 days (interquartile range [IQR], 3 to 7) in the camostat group and 5 days (IQR, 2 to 10) in the placebo group (P = 0·31). The hazard ratio for 30-day mortality in the camostat compared with the placebo group was 0·82 (95% confidence interval [CI], 0·24 to 2·79; P = 0·75). The frequency of adverse events was similar in the two groups. Median change in viral load from baseline to day 5 in the camostat group was -0·22 log10 copies/mL (p <0·05) and -0·82 log10 in the placebo group (P <0·05). INTERPRETATION: Under this protocol, camostat mesilate treatment was not associated with increased adverse events during hospitalization for Covid-19 and did not affect time to clinical improvement, progression to ICU admission or mortality. ClinicalTrials.gov Identifier: NCT04321096. EudraCT Number: 2020-001200-42.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.

Camostat mesylate against SARS-CoV-2 and COVID-19-Rationale, dosing and safety.

The coronavirus responsible for COVID-19, SARS-CoV-2, utilizes a viral membrane spike protein for host cell entry. For the virus to engage in host membrane fusion, SARS-CoV-2 utilizes the human transmembrane surface protease, TMPRSS2, to cleave and activate the spike protein. Camostat mesylate, an orally available well-known serine protease inhibitor, is a potent inhibitor of TMPRSS2 and has been hypothesized as a potential antiviral drug against COVID-19. In vitro human cell and animal studies have shown that camostat mesylate inhibits virus-cell membrane fusion and hence viral replication. In mice, camostat mesylate treatment during acute infection with influenza, also dependent on TMPRSS2, leads to a reduced viral load. The decreased viral load may be associated with an improved patient outcome. Because camostat mesylate is administered as an oral drug, it may be used in outpatients as well as inpatients at all disease stages of SARS-CoV-2 infection if it is shown to be an effective antiviral agent. Clinical trials are currently ongoing to test whether this well-known drug could be repurposed and utilized to combat the current pandemic. In the following, we will review current knowledge on camostat mesylate mode of action, potential benefits as an antiviral agent and ongoing clinical trials.

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.