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The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remain elusive. Leveraging whole-cell proteomics, we determined host signaling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signaling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signaling pathways causative for the differential pathogenicity of SARS-CoV-2 variants.
The highly transmissible Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in late 2021. Initial Omicron waves were primarily made up of sub-lineages BA.1 and/or BA.2, BA.4, and BA.5 subsequently became dominant in mid-2022, and several descendants of these sub-lineages have since emerged. Omicron infections have generally caused less severe disease on average than those caused by earlier variants of concern in healthy adult populations, at least, in part, due to increased population immunity. Nevertheless, healthcare systems in many countries, particularly those with low population immunity, have been overwhelmed by unprecedented surges in disease prevalence during Omicron waves. Pediatric admissions were also higher during Omicron waves compared with waves of previous variants of concern. All Omicron sub-lineages exhibit partial escape from wild-type (Wuhan-Hu 1) spike-based vaccine-elicited neutralizing antibodies, with sub-lineages with more enhanced immuno-evasive properties emerging over time. Evaluating vaccine effectiveness (VE) against Omicron sub-lineages has become challenging against a complex background of varying vaccine coverage, vaccine platforms, prior infection rates, and hybrid immunity. Original messenger RNA vaccine booster doses substantially improved VE against BA.1 or BA.2 symptomatic disease. However, protection against symptomatic disease waned, with reductions detected from 2 months after booster administration. While original vaccine-elicited CD8+ and CD4+ T-cell responses cross-recognize Omicron sub-lineages, thereby retaining protection against severe outcomes, variant-adapted vaccines are required to expand the breadth of B-cell responses and improve durability of protection. Variant-adapted vaccines were rolled out in late 2022 to increase overall protection against symptomatic and severe infections caused by Omicron sub-lineages and antigenically aligned variants with enhanced immune escape mechanisms.
A Corrigendum on "SARS-CoV-2 Omicron variants: burden of disease, impact on vaccine effectiveness and need for variant-adapted vaccines" by Pather S, Madhi SA, Cowling BJ, Moss P, Kamil JP, Ciesek S, Muik A and Türeci Ö (2023). . 14:1130539. doi: 10.3389/fimmu.2023.1130539
Omicron is the evolutionarily most distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) to date. We report that Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals resulted in strong neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 VOCs but not against the Omicron sublineages BA.4 and BA.5. BA.1 breakthrough infection induced a robust recall response, primarily expanding memory B (BMEM) cells against epitopes shared broadly among variants, rather than inducing BA.1-specific B cells. The vaccination-imprinted BMEM cell pool had sufficient plasticity to be remodeled by heterologous SARS-CoV-2 spike glycoprotein exposure. Whereas selective amplification of BMEM cells recognizing shared epitopes allows for effective neutralization of most variants that evade previously established immunity, susceptibility to escape by variants that acquire alterations at hitherto conserved sites may be heightened.
Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens.
Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity.
Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed.
Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.
Background: In recent months, Omicron variants of SARS-CoV-2 have become dominant in many regions of the world, and case numbers with Omicron subvariants BA.1 and BA.2 continue to increase. Due to numerous mutations in the spike protein, the efficacy of currently available vaccines, which are based on Wuhan-Hu 1 isolate of SARS-CoV-2, is reduced, leading to breakthrough infections. Efficacy of monoclonal antibody therapy is also likely impaired.
Methods: In our in vitro study using A549-AT cells constitutively expressing ACE2 and TMPRSS2, we determined and compared the neutralizing capacity of vaccine-elicited sera, convalescent sera and monoclonal antibodies against authentic SARS-CoV-2 Omicron BA.1 and BA.2 compared with Delta.
Findings: Almost no neutralisation of Omicron BA.1 and BA.2 was observed using sera from individuals vaccinated with two doses 6 months earlier, regardless of the type of vaccine taken. Shortly after the booster dose, most sera from triple BNT162b2-vaccinated individuals were able to neutralise both Omicron variants. In line with waning antibody levels three months after the booster, only weak residual neutralisation was observed for BA.1 (26%, n = 34, 0 median NT50) and BA.2 (44%, n = 34, 0 median NT50). In addition, BA.1 but not BA.2 was resistant to the neutralising monoclonal antibodies casirivimab/imdevimab, while BA.2 exhibited almost a complete evasion from the neutralisation induced by sotrovimab.
Interpretation: Both SARS-CoV-2 Omicron subvariants BA.1 and BA.2 escape antibody-mediated neutralisation elicited by vaccination, previous infection with SARS-CoV-2, and monoclonal antibodies. Waning immunity renders the majority of tested sera obtained three months after booster vaccination negative in BA.1 and BA.2 neutralisation. Omicron subvariant specific resistance to the monoclonal antibodies casirivimab/imdevimab and sotrovimab emphasizes the importance of genotype-surveillance and guided application.
Funding: This study was supported in part by the Goethe-Corona-Fund of the Goethe University Frankfurt (M.W.) and the Federal Ministry of Education and Research (COVIDready; grant 02WRS1621C (M.W.).
The SARS-CoV-2 Omicron variant is currently causing a large number of infections in many countries. A number of antiviral agents are approved or in clinical testing for the treatment of COVID-19. Despite the high number of mutations in the Omicron variant, we here show that Omicron isolates display similar sensitivity to eight of the most important anti-SARS-CoV-2 drugs and drug candidates (including remdesivir, molnupiravir, and PF-07321332, the active compound in paxlovid), which is of timely relevance for the treatment of the increasing number of Omicron patients. Most importantly, we also found that the Omicron variant displays a reduced capability of antagonising the host cell interferon response. This provides a potential mechanistic explanation for the clinically observed reduced pathogenicity of Omicron variant viruses compared to Delta variant viruses.
Recently, we have shown that SARS-CoV-2 Omicron virus isolates are less effective at inhibiting the host cell interferon response than Delta viruses. Here, we present further evidence that reduced interferon-antagonising activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Most importantly, we here also show that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapy candidates for Omicron patients, in particular in combination with other antiviral agents.
The new variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Omicron (B.1.1.529), is genetically very different from other VOCs. We compared Omicron with the preceding VOC Delta (B.1.617.2) and the wildtype strain (B.1) with respect to their interactions with the antiviral type I interferon (IFN-alpha/beta) response in infected cells. Our data indicate that Omicron has gained an elevated capability to suppress IFN-beta induction upon infection and to better withstand the antiviral state imposed by exogenously added IFN-alpha.
Background & Aims: Elimination of chronic HBV/HDV infection remains a major global health challenge. Targeting excessive hepatitis B surface antigen (HBsAg) release may provide an interesting window of opportunity to break immune tolerance and to achieve a functional cure using additional antivirals.
Methods: We evaluated a HBsAg-specific human monoclonal antibody, as part of either a prophylactic or therapeutic strategy, against HBV/HDV infection in cell culture models and in human-liver chimeric mice. To assess prophylactic efficacy, mice were passively immunized prior to infection with HBV or HBV/HDV (coinfection and superinfection setting). Therapeutic efficacy was assessed in HBV and HBV/HDV-coinfected mice receiving 4 weeks of treatment. Viral parameters (HBV DNA, HDV RNA and HBsAg) were assessed in mouse plasma.
Results: The antibody could effectively prevent HBV/HDV infection in a dose-dependent manner with IC50 values of ∼3.5 ng/ml. Passive immunization showed complete protection of mice from both HBV and HBV/HDV coinfection. Moreover, HDV superinfection was either completely prevented or at least attenuated in HBV-infected mice. Finally, antibody treatment in mice with established HBV/HDV infection resulted in a significant decline in viremia and a concomitant drop in on-treatment HBsAg, with a moderate viral rebound following treatment cessation.
Conclusion: We present data on a valuable antibody candidate that could complement other antivirals in strategies aimed at achieving functional cure of chronic HBV and HDV infection.
Impact and implications: Patients chronically infected with HBV may eventually develop liver cancer and are at great risk of being superinfected with HDV, which worsens and accelerates disease progression. Unfortunately, current treatments can rarely eliminate both viruses from chronically infected patients. In this study, we present data on a novel antibody that is able to prevent chronic HBV/HDV infection in a mouse model with a humanized liver. Moreover, antibody treatment of HBV/HDV-infected mice strongly diminishes viral loads during therapy. This antibody is a valuable candidate for further clinical development.
Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. Omicron sub-variants BA.4/5 have spread globally displacing the predeceasing variants. Due to the severe transmissibility and immune escape potential of BA.4/5, early monitoring was required to asses and implement countermeasures in time.
In this study, we monitored the prevalence of SARS-CoV-2 BA.4/5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North-Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability to detect BA.4/5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022 and the presence of BA.4/5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V. Finally, the mutant fractions were quantitatively monitored by digital PCR confirming BA.4/5 as the majority variant by 5th June 2022.
In conclusions, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variant spreading independent of individual test capacities.
Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19.
As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging–based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti–SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.
Vaccines are central to controlling the coronavirus disease 2019 (COVID-19) pandemic but the durability of protection is limited for currently approved COVID-19 vaccines. Further, the emergence of variants of concern (VoCs) that evade immune recognition has reduced vaccine effectiveness, compounding the problem. Here, we show that a single dose of a murine cytomegalovirus (MCMV)-based vaccine, which expresses the spike (S) protein of the virus circulating early in the pandemic (MCMVS), protects highly susceptible K18-hACE2 mice from clinical symptoms and death upon challenge with a lethal dose of D614G SARS-CoV-2. Moreover, MCMVS vaccination controlled two immune-evading VoCs, the Beta (B.1.135) and the Omicron (BA.1) variants in BALB/c mice, and S-specific immunity was maintained for at least 5 months after immunization, where neutralizing titers against all tested VoCs were higher at 5-months than at 1-month post-vaccination. Thus, cytomegalovirus (CMV)-based vector vaccines might allow for long-term protection against COVID-19.
Background: International travel poses the risk of importing SARS-CoV-2 infections and introducing new viral variants into the country of destination. Established measures include mandatory quarantine with the opportunity to abbreviate it with a negative rapid antigen test (RAT).
Methods: A total of 1,488 returnees were tested for SARS-CoV-2 with both PCR and RAT no earlier than 5 days after arrival. We assessed the sensitivity and specificity of the RAT. Positive samples were evaluated for infectivity in vitro in a cell culture outgrowth assay. We tracked if participants who tested negative were reported positive within 2 weeks of the initial test.
Results: Potential infectiousness was determined based on symptom onset analysis, resulting in a sensitivity of the antigen test of 89% in terms of infectivity. The specificity was 100%. All positive outgrowth assays were preceded by a positive RAT, indicating that all participants with proven in vitro infectivity were correctly identified. None of the negative participants tested positive during the follow-up.
Conclusions: RAT no earlier than the 5th day after arrival was a reliable method for detecting infectious travellers and can be recommended as an appropriate method for managing SARS-CoV-2 travel restrictions. Compliance to the regulations and a high standard of test quality must be ensured.
Objectives: Regarding reactogenicity and immunogenicity, heterologous COVID-19 vaccination regimens are considered as an alternative to conventional immunization schemes.
Methods: Individuals receiving either heterologous (ChAdOx1-S [AstraZeneca, Cambridge, UK]/BNT162b2 [Pfizer-BioNTech, Mainz, Germany]; n = 306) or homologous (messenger RNA [mRNA]-1273 [Moderna, Cambridge, Massachusetts, USA]; n = 139) vaccination were asked to participate when receiving their second dose. Reactogenicity was assessed after 1 month, immunogenicity after 1, 3, and/or 6 months, including a third dose, through SARS-CoV-2 antispike immunoglobulin G, surrogate virus neutralization test, and a plaque reduction neutralization test against the Delta (B.1.167.2) and Omicron (B.1.1.529; BA.1) variants of concern.
Results: The overall reactogenicity was lower after heterologous vaccination. In both cohorts, SARS-CoV-2 antispike immunoglobulin G concentrations waned over time with the heterologous vaccination demonstrating higher neutralizing activity than homologous mRNA vaccination after 3 months to low neutralizing levels in the Delta plaque reduction neutralization test after 6 months. At this point, 3.2% of the heterologous and 11.4% of the homologous cohort yielded low neutralizing activity against Omicron. After a third dose of an mRNA vaccine, ≥99% of vaccinees demonstrated positive neutralizing activity against Delta. Depending on the vaccination scheme and against Omicron, 60% to 87.5% of vaccinees demonstrated positive neutralizing activity.
Conclusion: ChAdOx1-S/BNT162b2 vaccination demonstrated an acceptable reactogenicity and immunogenicity profile. A third dose of an mRNA vaccine is necessary to maintain neutralizing activity against SARS-CoV-2. However, variants of concern-adapted versions of the vaccines would be desirable.
Omicron BA.1 variant isolates were previously shown to replicate less effectively in interferon-competent cells and to be more sensitive to interferon treatment than a Delta isolate. Here, an Omicron BA.2 isolate displayed intermediate replication patterns in interferon-competent Caco-2-F03 cells when compared to BA.1 and Delta isolates. Moreover, BA.2 was less sensitive than BA.1 and similarly sensitive as Delta to betaferon treatment. Delta and BA.1 displayed similar sensitivity to the approved anti-SARS-CoV-2 drugs remdesivir, nirmatrelvir, EIDD-1931 (the active metabolite of molnupiravir) and the protease inhibitor aprotinin, whereas BA.2 was less sensitive than Delta and BA.1 to EIDD-1931, nirmatrelvir and aprotinin. Nirmatrelvir, EIDD-1931, and aprotinin (but not remdesivir) exerted synergistic antiviral activity in combination with betaferon, with some differences in the extent of synergism detected between the different SARS-CoV-2 variants. In conclusion, even closely related SARS-CoV-2 (sub)variants can differ in their biology and in their response to antiviral treatments. Betaferon combinations with nirmatrelvir and, in particular, with EIDD-1931 and aprotinin displayed high levels of synergism, which makes them strong candidates for clinical testing. Notably, effective antiviral combination therapies are desirable, as a higher efficacy is expected to reduce resistance formation.