A computationally designed ACE2 decoy has broad efficacy against SARS-CoV-2 omicron variants and related viruses in vitro and in vivo.

SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a 'decoy' to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy, FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments. FLIF displayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) against FLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys like FLIF may be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants.

SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response.

Reports of post-acute COVID-19 syndrome, in which the inflammatory response persists even after SARS-CoV-2 has disappeared, are increasing1, but the underlying mechanisms of post-acute COVID-19 syndrome remain unknown. Here, we show that SARS-CoV-2-infected cells trigger senescence-like cell-cycle arrest2,3 in neighboring uninfected cells in a paracrine manner via virus-induced cytokine production. In cultured human cells or bronchial organoids, these SASR-CoV-2 infection-induced senescent cells express high levels of a series of inflammatory factors known as senescence-associated secretory phenotypes (SASPs)4 in a sustained manner, even after SARS-CoV-2 is no longer detectable. We also show that the expression of the senescence marker CDKN2A (refs. 5,6) and various SASP factor4 genes is increased in the pulmonary cells of patients with severe post-acute COVID-19 syndrome. Furthermore, we find that mice exposed to a mouse-adapted strain of SARS-CoV-2 exhibit prolonged signs of cellular senescence and SASP in the lung at 14 days after infection when the virus was undetectable, which could be substantially reduced by the administration of senolytic drugs7. The sustained infection-induced paracrine senescence described here may be involved in the long-term inflammation caused by SARS-CoV-2 infection.

Psychological distress among healthcare providers in oncology during the COVID-19 pandemic in Japan: The mediating role of moral distress and resilience.

Objective: Even though vaccines have become widespread, there is an explosion of infection due to the emergence of new mutant strains, and support for healthcare providers' mental health is necessary. The aims of this study were to explore factors associated with the psychological distress, and to determine the degree of association between moral distress, resilience and psychological distress in order to consider intervention models for psychological distress of healthcare providers involved with cancer patients during the COVID-19 pandemic. Method: We conducted a cross-sectional survey among healthcare providers at the National Cancer Center, Japan. Psychological distress was assessed by the Hospital Anxiety and Depression Scale. We also assessed moral distress using the Moral Distress Thermometer and resilience using the Connor-Davidson Resilience Scale 10 in April and May 2020 which was the first surge of the epidemic period. Results: Five hundred sixty-six of 3,900 healthcare providers (14.5%) responded. Sixty-eight percent (385/566) responders were above the Hospital Anxiety and Depression Scale cutoff. Hierarchical regression analyses indicated that nurses, allied health professionals and office workers/engineers (odds ratio = 4.63; 95% confidence interval 1.90-11.29; p < 0.001, odds ratio = 3.88; 95% confidence interval 1.88-8.00; p < 0.001, odds ratio = 2.10; 95% confidence interval 1.06-4.18; p < 0.05) as well as healthcare providers with low resilience (odds ratio = 0.88; 95% confidence interval 0.85-0.91; p < 0.001) were at risk of psychological distress. Moral distress was not significantly associated with prevalence of psychological distress. Conclusion: During the first surge of the pandemic, a high prevalence of psychological distress was demonstrated among cancer center healthcare providers. The study suggests that mental health care should be available to cancer care providers. Since the COVID-19 pandemic is still going on, in addition to the efforts by our facilities, we should consider interventions that promote resilience and a feasibility study of these interventions.

SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses.

Many patients with severe COVID-19 suffer from pneumonia and the elucidation of the mechanisms underlying the development of this severe condition is important. The in vivo function of the ORF8 protein secreted by SARS-CoV-2 is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2 and found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrated that the serum ORF8 protein levels are well-correlated with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a SARS-CoV-2 viral cytokine involved in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

Stimulation of interferon-ß responses by aberrant SARS-CoV-2 small viral RNAs acting as retinoic acid-inducible gene-I agonists.

Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5' ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5' end svRNAs are RIG-I agonists and induce the IFN-ß response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5'-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5' end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

SARS-CoV-2 ORF6 disrupts nucleocytoplasmic trafficking to advance viral replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin ß1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-ß but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-ß. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Diagnostic Performance of Computed Tomography Imaging for COVID-19 in a Region with Low Disease Prevalence.

Coronavirus disease 2019 (COVID-19) was first reported in Wuhan, China, in December 2019 as an outbreak of pneumonia of unknown origin. Previous studies have suggested the utility of chest computed tomography (CT) in the diagnosis of COVID-19 because of its high sensitivity (93%-97%), relatively simple procedure, and rapid test results. This study, performed in Japan early in the epidemic when COVID-19 prevalence was low, evaluated the diagnostic accuracy of chest CT in a population presenting with lung diseases having CT findings similar to those of COVID-19. We retrospectively included all consecutive patients (≥18 years old) presenting to the outpatient department of Keio University Hospital between March 1 and May 31, 2020, with fever and respiratory symptoms. We evaluated the performance of diagnostic CT for COVID-19 by using polymerase chain reaction (PCR) results as the reference standard. We determined the numbers of false-positive (FP) results and assessed the clinical utility using decision curve analysis. Of the 175 patients, 22 were PCR-positive. CT had a sensitivity of 68% and a specificity of 57%. Patients with FP results on CT diagnosis were mainly diagnosed with diseases mimicking COVID-19, e.g., interstitial lung disease. Decision curve analysis indicated that the clinical utility of CT imaging was limited. The diagnostic performance of CT for COVID-19 was inadequate in an area with low COVID-19 prevalence and a high prevalence of other lung diseases with chest CT findings similar to those of COVID-19. Considering this insufficient diagnostic performance, CT findings should be evaluated in the context of additional medical information to diagnose COVID-19.

[Advances in the reverse genetics system for RNA viruses].

RNA viruses are responsible for several infectious diseases, including dengue fever, Zika fever, and COVID-19. Reverse genetics is a powerful tool to elucidate which domain or mutations in RNA viruses determine their pathogenicity and ability to evade antiviral drugs and host immune response. Previous reverse genetics systems for flaviviruses and coronaviruses have been technically challenging and time-consuming, thereby hampering the further understanding of events during viral evolution. A novel reverse genetics system-circular polymerase extension reaction (CPER)-has been developed to overcome this limitation. CPER is based on PCR-mediated assembly of DNA fragments that encode the whole genome of these viruses. CPER requires a relatively short time to introduce specific mutations into the viral genome of flaviviruses and SARS-CoV-2. In this review article, we explain the mode of action of this system and discuss the future direction of reverse genetics for RNA viruses.

Preparing for the Next Pandemic: An Asian National Cancer Centers Alliance (ANCCA) Initiative.

The COVID-pandemic has shown significant impact on cancer care from early detection, management plan to clinical outcomes of cancer patients. The Asian National Cancer Centres Alliance (ANCCA) has put together the 9 "Ps" as guidelines for cancer programs to better prepare for the next pandemic. The 9 "Ps" are Priority, Protocols and Processes, Patients, People, Personal Protective Equipments (PPEs), Pharmaceuticals, Places, Preparedness, and Politics. Priority: to maintain cancer care as a key priority in the health system response even during a global infectious disease pandemic. Protocol and processes: to develop a set of Standard Operating Procedures (SOPs) and have relevant expertise to man the Disease Outbreak Response (DORS) Taskforce before an outbreak. Patients: to prioritize patient safety in the event of an outbreak and the need to reschedule cancer management plan, supported by tele-consultation and use of artificial intelligence technology. People: to have business continuity planning to support surge capacity. PPEs and Pharmaceuticals: to develop plan for stockpiles management, build local manufacturing capacity and disseminate information on proper use and reduce wastage. Places: to design and build cancer care facilities to cater for the need of triaging, infection control, isolation and segregation. Preparedness: to invest early on manpower building and technology innovations through multisectoral and international collaborations. Politics: to ensure leadership which bring trust, cohesion and solidarity for successful response to pandemic and mitigate negative impact on the healthcare system.

Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

The Impact of COVID-19 on Cancer Care in the Post Pandemic World: Five Major Lessons Learnt from Challenges and Countermeasures of Major Asian Cancer Centres.

OBJECTIVE: The COVID-19 pandemic has dramatically affected healthcare services around Asia. The Asian National Cancer Centres Alliance and the Asia-Pacific Organisation for Cancer Prevention collaborated to assess the mid- and long- term impact of COVID-19 to cancer care in Asia. METHODS: The two entities organised a combined symposium and post-meeting interactions among representatives of major cancer centres from seventeen Asian countries to outlining major challenges and countermeasures. RESULTS: Participating stakeholders distilled five big questions. 1) "Will there be an explosion of late-stage cancers after the pandemic?" To address and recover from perceived delayed prevention, screening, treatment and care challenges, collaboration of key stakeholders in the region and alignment in cancer care management, policy intervention and cancer registry initiatives would be of essential value. 2) "Operations and Finance" The pandemic has resulted in significant material and financial casualties. Flagged acute challenges (shortages of supplies, imposition of lockdown) as well as longer-standing reduction of financial revenue, manpower, international collaboration, and training should also be addressed. 3) "Will telemedicine and technological innovations revolutionize cancer care?" Deploying and implementing telemedicine such as teleconsultation and virtual tumour boards were considered invaluable. These innovations could become a new regular practice, leading to expansion of tele-collaboration through collaboration of institutions in the region. 4) "Will virtual conferences continue after the pandemic?" Virtual conferences during the pandemic have opened new doors for knowledge sharing, especially for representatives of low- and middle-income countries in the region, while saving time and costs of travel. 5) "How do we prepare for the next pandemic or international emergency?" Roadmaps for action to improve access to appropriate patient care and research were identified and scrutinised. CONCLUSION: Through addressing these five big questions, focused collaboration among members and with international organisations such as City Cancer Challenge will allow enhanced preparedness for future international emergencies.
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COVID-19 pneumonia in a patient with adult T-cell leukemia-lymphoma.

Although some patients with COVID-19 develop only mild symptoms, fatal complications have been observed among those with comorbidities. As patients with cancer are immunocompromised, they are thought to have a high risk of severe illness associated with COVID-19. We report a COVID-19 patient with adult T-cell leukemia-lymphoma (ATL) who was treated using favipiravir. A 69-year-old woman with lymphoma-type ATL was treated using cyclophosphamide, doxorubicin, vincristine, prednisolone and mogamulizumab (M-CHOP) with substantial efficacy. However, in cycle 4 of M-CHOP therapy, she developed fever with mild cough. The patient was admitted to the hospital and CT revealed bilateral ground-glass opacities. SARS-CoV-2 was detected by RT-PCR and the patient was diagnosed with COVID-19. Considering severe immunosuppression caused by ATL, we initiated favipiravir therapy. Subsequently, the fever improved without antipyretics and her C-reactive protein level decreased rapidly. SARS-CoV-2 PCR tests were negative on days 17 and 18 of favipiravir therapy, and the patient was discharged without residual disease on the final CT. This is the first documented case of COVID-19 in a patient with ATL. Although severe immunosuppression caused by ATL was present, severe COVID-19 pneumonia did not develop. The immunosuppressed condition caused by hematological malignancy may not always be a risk factor for severe illness associated with COVID-19. Further accumulation of data regarding COVID-19 in patients with hematological malignancies is warranted to clarify the risk factors for severe illness, the best-in-class antiviral agent, and the optimal treatment strategy in this population.