Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.

Establishment of Recombinant Trisegmented Mopeia Virus Expressing Two Reporter Genes for Screening of Mammarenavirus Inhibitors.

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions.

Advanced Molecular Tweezers with Lipid Anchors against SARS-CoV-2 and Other Respiratory Viruses.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

Unterdrückung der SARS‐CoV‐2‐Replikation mit stabilisierten und durch Klick‐Chemie modifizierten siRNAs

Die Entstehung von leichter übertragbaren oder aggressiveren Varianten von SARS‐CoV‐2 erfordert die Entwicklung von antiviralen Medikamenten, die schnell an sich entwickelnde virale Escape‐Mutationen anpassbar sind. Hier berichten wir über die Synthese von chemisch stabilisierter small interfering RNA (siRNA) gegen SARS‐CoV‐2. Die siRNA kann mit Hilfe von CuI‐katalysierter Klick‐Chemie mit Rezeptorliganden wie Peptiden zusätzlich modifiziert werden. Wir zeigen, dass optimierte siRNAs die Viruslast und die virus‐induzierte Zytotoxizität in Zelllinien, die mit SARS‐CoV‐2 infiziert sind, um bis zu fünf Größenordnungen reduzieren können. Darüber hinaus zeigen wir, dass eine mit einem ACE2‐bindenden Peptid‐konjugierte siRNA in der Lage ist, die Virusreplikation und die virus‐induzierte Apoptose in mukoziliären 3D‐Lungenmikrogeweben zu reduzieren. Eine Änderung der siRNA‐Sequenz ermöglicht eine schnelle Anpassung ihrer antiviralen Aktivität gegen verschiedene Virusvarianten. Die Möglichkeit, die siRNA mittels Klick‐Chemie an Rezeptorliganden zu konjugieren, erleichtert die Entwicklung zielgerichteter siRNAs für eine flexible antivirale Abwehrstrategie.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

RIG-I-induced innate antiviral immunity protects mice from lethal SARS-CoV-2 infection.

The SARS-CoV-2 pandemic has underscored the need for rapidly usable prophylactic and antiviral treatments against emerging viruses. The targeted stimulation of antiviral innate immune receptors can trigger a broad antiviral response that also acts against new, unknown viruses. Here, we used the K18-hACE2 mouse model of COVID-19 to examine whether activation of the antiviral RNA receptor RIG-I protects mice from lethal SARS-CoV-2 infection and reduces disease severity. We found that prophylactic, systemic treatment of mice with the specific RIG-I ligand 3pRNA, but not type I interferon, 1-7 days before viral challenge, improved survival of mice by up to 50%. Survival was also improved with therapeutic 3pRNA treatment starting 1 day after viral challenge. This improved outcome was associated with lower viral load in oropharyngeal swabs and in the lungs and brains of 3pRNA-treated mice. Moreover, 3pRNA-treated mice exhibited reduced lung inflammation and developed a SARS-CoV-2-specific neutralizing antibody response. These results demonstrate that systemic RIG-I activation by therapeutic RNA oligonucleotide agonists is a promising strategy to convey effective, short-term antiviral protection against SARS-CoV-2 infection, and it has great potential as a broad-spectrum approach to constrain the spread of newly emerging viruses until virus-specific therapies and vaccines become available.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

Correlation between a quantitative anti-SARS-CoV-2 IgG ELISA and neutralization activity.

In the current COVID-19 pandemic, a better understanding of the relationship between merely binding and functionally neutralizing antibodies is necessary to characterize protective antiviral immunity following infection or vaccination. This study analyzes the level of correlation between the novel quantitative EUROIMMUN Anti-SARS-CoV-2 QuantiVac ELISA (IgG) and a microneutralization assay. A panel of 123 plasma samples from a COVID-19 outbreak study population, preselected by semiquantitative anti-SARS-CoV-2 IgG testing, was used to assess the relationship between the novel quantitative ELISA (IgG) and a microneutralization assay. Binding IgG targeting the S1 antigen was detected in 106 (86.2%) samples using the QuantiVac ELISA, while 89 (72.4%) samples showed neutralizing antibody activity. Spearman's correlation analysis demonstrated a strong positive relationship between anti-S1 IgG levels and neutralizing antibody titers (rs = 0.819, p < 0.0001). High and low anti-S1 IgG levels were associated with a positive predictive value of 72.0% for high-titer neutralizing antibodies and a negative predictive value of 90.8% for low-titer neutralizing antibodies, respectively. These results substantiate the implementation of the QuantiVac ELISA to assess protective immunity following infection or vaccination.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Infection fatality rate of SARS-CoV2 in a super-spreading event in Germany.

A SARS-CoV2 super-spreading event occurred during carnival in a small town in Germany. Due to the rapidly imposed lockdown and its relatively closed community, this town was seen as an ideal model to investigate the infection fatality rate (IFR). Here, a 7-day seroepidemiological observational study was performed to collect information and biomaterials from a random, household-based study population. The number of infections was determined by IgG analyses and PCR testing. We found that of the 919 individuals with evaluable infection status, 15.5% (95% CI:[12.3%; 19.0%]) were infected. This is a fivefold higher rate than the reported cases for this community (3.1%). 22.2% of all infected individuals were asymptomatic. The estimated IFR was 0.36% (95% CI:[0.29%; 0.45%]) for the community and 0.35% [0.28%; 0.45%] when age-standardized to the population of the community. Participation in carnival increased both infection rate (21.3% versus 9.5%, p < 0.001) and number of symptoms (estimated relative mean increase 1.6, p = 0.007). While the infection rate here is not representative for Germany, the IFR is useful to estimate the consequences of the pandemic in places with similar healthcare systems and population characteristics. Whether the super-spreading event not only increases the infection rate but also affects the IFR requires further investigation.