ABSTRACT
Remdesivir was the first drug to be approved for the treatment of severe COVID-19; followed by molnupiravir (another prodrug of a nucleoside analogue) and the protease inhibitor nirmatrelvir. Combination of antiviral drugs may result in improved potency and help to avoid or delay the development of resistant variants. We set out to explore the combined antiviral potency of GS-441524 (the parent nucleoside of remdesivir) and molnupiravir against SARS-CoV-2. In SARS-CoV-2 (BA.5) infected A549-Dual hACE2-TMPRSS2 cells, the combination resulted in an overall additive antiviral effect with a synergism at certain concentrations. Next, the combined effect was explored in Syrian hamsters infected with SARS-CoV-2 (Beta, B.1.351); treatment was started at the time of infection and continued twice daily for four consecutive days. At 4 day 4 post-infection, GS-441524 (50 mg/kg, oral BID) and molnupiravir (150 mg/kg, oral BID) as monotherapy reduced infectious viral loads by 0.5 and 1.6 log10, respectively, compared to the vehicle control. When GS-441524 (50 mg/kg, BID) and molnupiravir (150 mg/kg, BID) were combined, infectious virus was no longer detectable in the lungs of 7 out of 10 of the treated hamsters (4.0 log10 reduction) and titers in the other animals were reduced by ~2 log10. The combined antiviral activity of molnupiravir which acts by inducing lethal mutagenesis and GS-441524, which acts as a chain termination appears to be highly effective in reducing SARS-CoV-2 replication/infectivity. The unexpected potent antiviral effect of the combination warrants further exploration as a potential treatment for COVID-19.
Subject(s)
Severe Acute Respiratory Syndrome , Adenomatous Polyposis Coli , COVID-19ABSTRACT
The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic target for the treatment of COVID-19. Nirmatrelvir is the only the 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization; other 3Lpro inhibitors are in development. We recently repored on the in vitro selection of a SARS-CoV2 3CLpro (L50F-E166A-L167F; short 3CLprores) virus that is cross-resistant with nirmatrelvir and yet other 3CLpro inhibitors. Here, we demonstrate that the resistant virus replicates efficiently in the lungs of intranassaly infected hamsters and that it causes a lung pathology that is comparable to that caused by the WT virus. Moreover, 3CLprores infected hamsters transmit the virus efficiently to co-housed non-infected contact hamsters. Fortunately, resistance to Nirmatrelvir does not readily develop (in the clinical setting) since the drug has a relatively high barrier to resistance. Yet, as we demonstrate, in case resistant viruses emerge, they may easily spread and impact therapeutic options for others. Therefore, the use of SARS-CoV-2 3CLpro protease inhibitors in combinations with drugs that have a different mechanism of action, may be considered to avoid the development of drug-resistant viruses in the future.
Subject(s)
COVID-19ABSTRACT
Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2.
Subject(s)
COVID-19 , X-Linked Combined Immunodeficiency DiseasesABSTRACT
Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing 2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.
Subject(s)
Coronavirus InfectionsABSTRACT
Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.
Subject(s)
COVID-19ABSTRACT
The emergence of SARS-CoV-2 variants of concern (VoCs) has exacerbated the COVID-19 pandemic. End of November 2021, a new SARS-CoV-2 variant namely the omicron (B.1.1.529) emerged. Since this omicron variant is heavily mutated in the spike protein, WHO classified this variant as the 5th variant of concern (VoC). We previously demonstrated that the other SARS-CoV-2 VoCs replicate efficiently in Syrian hamsters, alike also the ancestral strains. We here wanted to explore the infectivity of the omicron variant in comparison to the ancestral D614G strain. Strikingly, in hamsters that had been infected with the omicron variant, a 3 log10 lower viral RNA load was detected in the lungs as compared to animals infected with D614G and no infectious virus was detectable in this organ. Moreover, histopathological examination of the lungs from omicron-infecetd hamsters revealed no signs of peri-bronchial inflammation or bronchopneumonia. Further experiments are needed to determine whether the omicron VoC replicates possibly more efficiently in the upper respiratory tract of hamsters than in their lungs.
Subject(s)
Infections , Bronchopneumonia , COVID-19 , InflammationABSTRACT
Current licensed COVID-19 vaccines are based on antigen sequences of initial SARS-CoV-2 isolates that emerged in 2019. By mid 2021 these historical virus strains have been completely replaced by new cosmopolitan SARS-CoV-2 lineages. The ongoing pandemic has been further driven by emerging variants of concern (VOC) Alpha, Beta, Gamma and, lately predominant, Delta. These are characterized by an increased transmissibility and possible escape from naturally acquired or vaccine-induced immunity. We here show, using a YF17D-vectored first-generation COVID-19 vaccine (Sanchez-Felipe et al., 2021) and a stringent hamster challenge model (Abdelnabi et al., 2021) that the immunity elicited by a prototypic spike antigen is insufficient to provide optimal protection against the Beta VoC, urging for an antigenic update. We therefore designed an updated second-generation vaccine candidate that carries the sequence of a spike antigen that includes crucial epitopes from multiple VOCs. This vaccine candidate yielded a marked change in target antigen spectrum covered as demonstrated by (i) antigenic cartography and (ii) full protection against infection and virus-induced disease caused by any of the four VOCs (Alpha, Beta, Gamma and Delta) used for challenge. This more universal COVID-19 vaccine candidate also efficiently blocked direct transmission of VOC Delta from vaccinated infected hamsters to non-vaccinated sentinels under prolonged co-housing conditions. In conclusion, our data suggest that current first-generation COVID-19 vaccines need to be adapted to cover emerging sequence diversity of VOC to preserve vaccine efficacy and to contain virus spread at the community level.
Subject(s)
COVID-19ABSTRACT
There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the main viral protease (Mpro, 3CLpro) that can be dosed orally; the compound is in clinical development. We demonstrate that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels. The trough drug concentration at this efficacious dose were above the in vitro efficacious concentrations.
ABSTRACT
We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.
Subject(s)
Lung Diseases , COVID-19ABSTRACT
Within one year after its emergence, more than 108 million people contracted SARS-CoV-2 and almost 2.4 million succumbed to COVID-19. New SARS-CoV-2 variants of concern (VoC) are emerging all over the world, with the threat of being more readily transmitted, being more virulent, or escaping naturally acquired and vaccine-induced immunity. At least three major prototypic VoC have been identified, i.e. the UK (B.1.1.7), South African (B.1.351) and Brazilian (B.1.1.28.1), variants. These are replacing formerly dominant strains and sparking new COVID-19 epidemics and new spikes in excess mortality. We studied the effect of infection with prototypic VoC from both B.1.1.7 and B.1.351 lineages in Syrian golden hamsters to assess their relative infectivity and pathogenicity in direct comparison to two basal SARS-CoV-2 strains isolated in early 2020. A very efficient infection of the lower respiratory tract of hamsters by these VoC is observed. In line with clinical evidence from patients infected with these VoC, no major differences in disease outcome were observed as compared to the original strains as was quantified by (i) histological scoring, (ii) micro-computed tomography, and (iii) analysis of the expression profiles of selected antiviral and pro-inflammatory cytokine genes. Noteworthy however, in hamsters infected with VoC B.1.1.7, a particularly strong elevation of proinflammatory cytokines was detected. Overall, we established relevant preclinical infection models that will be pivotal to assess the efficacy of current and future vaccine(s) (candidates) as well as therapeutics (small molecules and antibodies) against two important SARS-CoV-2 VoC.
Subject(s)
COVID-19ABSTRACT
In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The FDA-approved HIV protease inhibitor Nelfinavir is one of the drugs that has been reported to inhibit in vitro SARS-CoV2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 mg/kg BID or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] did not reduce viral RNA loads nor infectious virus titres in the lungs (as compared to the vehicle control at the end of treatment) the drug markedly improved virus-induced lung pathology at doses that were well tolerated. Yet, a massive interstitial infiltration of neutrophils was observed in the lungs of treated (infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology that is unrelated to an antiviral effect warrants further exploration in the context of the treatment of COVID-19.
Subject(s)
COVID-19 , Severe Acute Respiratory Syndrome , InfectionsABSTRACT
Three pathogenic human coronaviruses have emerged, with SARS-2 causing a global pandemic. While therapeutic antibodies targeting the SARS-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report three antibodies binding epitopes conserved on the pre-fusion MERS, SARS-1 and SARS-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes in in vitro SARS-2 cell fusion and pseudovirus assays. Hydrogen-Deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, consistent with binding to natural and engineered spike variants. This location at the spike trimer interface suggests 3A3 prevents the S2 conformational rearrangements required for virus-host cell fusion. This work defines a highly conserved vulnerable site on the SARS-2 S2 domain and may help guide design of pan-protective spike immunogens.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 g of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 g or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-{gamma}, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.
Subject(s)
Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
BACKGROUND: There has been considerable speculation regarding the potential of PVP-I nasal disinfection as an adjunct to other countermeasures during the ongoing SARS-CoV-2 pandemic. Nasodine is a commercial formulation of 0.5% PVP-I that has been evaluated for safety and efficacy in human trials as a treatment for the common cold, including a Phase III trial (ANZCTR: ACTRN12619000764134). This study presents the first report of the in vitro efficacy of this formulation against SARS-CoV-2. METHODS: We conducted in vitro experiments to determine if the PVP-I formulation inactivated SARS-CoV-2 using two independent assays and virus isolates, and incorporating both PCR-based detection and cell culture methods to assess residual virus after exposure to the formulation. RESULTS: Based on cell culture results, the PVP-I formulation was found to rapidly inactivate SARS-CoV-2 isolates in vitro in short timeframes (15 seconds to 15 minutes) consistent with the minimum and maximum potential residence time in the nose. The Nasodine formula was found to be more effective than 0.5% PVP-I in saline. Importantly, it was found that the formulation inactivated culturable virus but had no effect on PCR-detectable viral RNA. CONCLUSIONS: The PVP-I formulation eliminated the viability of SARS-CoV-2 virus with short exposure times consistent with nasal use. PCR alone may not be adequate for viral quantification in nasal PVP-I studies; future studies should incorporate cell culture to assess viral viability. Nasal disinfection with PVP-I may be a useful intervention for newly-diagnosed COVID-19 patients to reduce transmission risk and disease progression to the lower respiratory tract.
Subject(s)
COVID-19ABSTRACT
SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed "Nanotraps", completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection.
Subject(s)
COVID-19ABSTRACT
Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.
Subject(s)
Coronavirus Infections , COVID-19 , Virus DiseasesABSTRACT
The novel {beta}-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 101 million people and resulted in 2.2 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provide evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is a master regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. These data, accordingly, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.
Subject(s)
Virus Diseases , COVID-19 , InflammationABSTRACT
Since its emergence in Wuhan, China in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide resulting in a global pandemic with >1.5 million deaths until now. In the search for small molecule inhibitors of SARS-CoV-2, drug repurposing is being extensively explored. Molnupiravir (EIDD-2801) is an orally bioavailable nucleoside analog that possesses a relatively broad-spectrum antiviral activity including against coronaviruses. We here studied the effect of EIDD-2801 in a well-established Syrian hamster SARS-CoV2 infection model. Treatment of SARS-CoV-2-infected hamsters with 200 mg/kg BID of EIDD-2801 for four consecutive days, starting from the day of infection, significantly reduced infectious virus titers and viral RNA loads in the lungs and markedly improved lung histopathology. When onset of treatment was delayed until 1 or 2 days after infection, a very modest antiviral effect was observed. The potential of EIDD-2801 for the treatment and or prevention of SARS-CoV2 deserves further attention.
Subject(s)
Coronavirus Infections , Severe Acute Respiratory SyndromeABSTRACT
Epidemiological and clinical reports have indicated that the host immune response to SARS-CoV-2, more so than viral factors, determines COVID-19 disease severity. To elucidate the immunopathology underlying COVID-19 severity, cytokine and multiplex immune profiling was performed in mild-moderate and critically-ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, and was more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels followed by deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presenting machinery was reduced in critical disease, while also neutrophils contributed to disease severity and local tissue damage by amplifying hypercytokinemia and neutrophil extracellular trap formation. We suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.