Potent neutralizing broad-spectrum antibody against SARS-CoV-2 generated from dual-antigen-specific B cells from convalescents.

Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing antibodies from B cells of convalescents, by using two types of receptor-binding domains, Wuhan strain and the Gamma variant as bait. From 172 antibodies generated, six antibodies neutralized all strains prior to the Omicron variant, and the five antibodies were able to neutralize some of the Omicron sub-strains. Structural analysis showed that these antibodies have a variety of characteristic binding modes, such as ACE2 mimicry. We subjected a representative antibody to the hamster infection model after introduction of the N297A modification, and observed a dose-dependent reduction of the lung viral titer, even at a dose of 2 mg/kg. These results demonstrated that our antibodies have certain antiviral activity as therapeutics, and highlighted the importance of initial cell-screening strategy for the efficient development of therapeutic antibodies.

In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters.

BACKGROUND: The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized. METHODS: We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo. FINDINGS: S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected. INTERPRETATION: Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity. FUNDING: A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Characterization of SARS-CoV-2 Omicron BA.2.75 clinical isolates.

The prevalence of the Omicron subvariant BA.2.75 rapidly increased in India and Nepal during the summer of 2022, and spread globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs is higher than that of BA.2 and BA.5. Of note, BA.2.75 causes focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which is not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicates better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 in a hamster model and should be closely monitored.

Plasminogen activator inhibitor 1 is not a major causative factor for exacerbation in a mouse model of SARS-CoV-2 infection.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global pandemic. Although several vaccines targeting SARS-CoV-2 spike proteins protect against COVID-19 infection, mutations affecting virus transmissibility and immune evasion potential have reduced their efficacy, leading to the need for a more efficient strategy. Available clinical evidence regarding COVID-19 suggests that endothelial dysfunction with thrombosis is a central pathogenesis of progression to systemic disease, in which overexpression of plasminogen activator inhibitor-1 (PAI-1) may be important. Here we developed a novel peptide vaccine against PAI-1 and evaluated its effect on lipopolysaccharide (LPS)-induced sepsis and SARS-CoV-2 infection in mice. Administration of LPS and mouse-adapted SARS-CoV-2 increased serum PAI-1 levels, although the latter showed smaller levels. In an LPS-induced sepsis model, mice immunized with PAI-1 vaccine showed reduced organ damage and microvascular thrombosis and improved survival compared with vehicle-treated mice. In plasma clot lysis assays, vaccination-induced serum IgG antibodies were fibrinolytic. However, in a SARS-CoV-2 infection model, survival and symptom severity (i.e., body weight reduction) did not differ between vaccine- and vehicle-treated groups. These results indicate that although PAI-1 may promote the severity of sepsis by increasing thrombus formation, it might not be a major contributor to COVID-19 exacerbation.

Co-administration of Favipiravir and the Remdesivir Metabolite GS-441524 Effectively Reduces SARS-CoV-2 Replication in the Lungs of the Syrian Hamster Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide since December 2019, causing coronavirus disease 2019 (COVID-19). Although vaccines for this virus have been developed rapidly, repurposing drugs approved to treat other diseases remains an invaluable treatment strategy. Here, we evaluated the inhibitory effects of drugs on SARS-CoV-2 replication in a hamster infection model and in in vitro assays. Favipiravir significantly suppressed virus replication in hamster lungs. Remdesivir inhibited virus replication in vitro, but was not effective in the hamster model. However, GS-441524, a metabolite of remdesivir, effectively suppressed virus replication in hamsters. Co-administration of favipiravir and GS-441524 more efficiently reduced virus load in hamster lungs than did single administration of either drug for both the prophylactic and therapeutic regimens; prophylactic co-administration also efficiently inhibited lung inflammation in the infected animals. Furthermore, pretreatment of hamsters with favipiravir and GS-441524 effectively protected them from virus transmission via respiratory droplets upon exposure to infected hamsters. Repurposing and co-administration of antiviral drugs may help combat COVID-19. IMPORTANCE During a pandemic, repurposing drugs that are approved for other diseases is a quick and realistic treatment option. In this study, we found that co-administration of favipiravir and the remdesivir metabolite GS-441524 more effectively blocked SARS-CoV-2 replication in the lungs of Syrian hamsters than either favipiravir or GS-441524 alone as part of a prophylactic or therapeutic regimen. Prophylactic co-administration also reduced the severity of lung inflammation. Moreover, co-administration of these drugs to naive hamsters efficiently protected them from airborne transmission of the virus from infected animals. Since both drugs are nucleotide analogs that interfere with the RNA-dependent RNA polymerases of many RNA viruses, these findings may also help encourage co-administration of antivirals to combat future pandemics.

SARS-CoV-2 Transmission from Virus-Infected Dead Hamsters.

Although it has been 2.5 years since the coronavirus disease 2019 (COVID-19) pandemic began, the transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a dead infected body remains unclear, and in Japan, bereaved family members are often not allowed to view in person a loved one who has died from COVID-19. In this study, we analyzed the possibility of SARS-CoV-2 transmission from a dead body using a hamster model. We also analyzed the effect of "angel care"--in which the pharynx, nostrils, and rectum are plugged--and embalming on reducing transmissibility from dead bodies. We found that SARS-CoV-2 could be transmitted from the bodies of animals that had died within a few days of infection; however, angel care and embalming were effective in preventing transmission from the dead bodies. These results suggest that protection from infection is essential when in contact with a SARS-CoV-2-infected dead body and that sealing the cavities of a dead body is an important infection control step if embalming is not performed. IMPORTANCE We found that SARS-CoV-2 could be transmitted from a dead body, presumably via postmortem gases. However, we also found that postmortem care, such as plugging the pharynx, nostrils, and rectum or embalming the corpse, could prevent transmission from the dead body. These results indicate that protection from infection is essential when handling infected corpses and that appropriate care of SARS-CoV-2-infected corpses is important.

Potent SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

Characterization of SARS-CoV-2 Omicron BA.4 and BA.5 isolates in rodents.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern1-4. Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral entry, raising concerns that the replication capacity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here we have evaluated the replicative ability and pathogenicity of BA.4 and BA.5 isolates in wild-type Syrian hamsters, human ACE2 (hACE2) transgenic hamsters and hACE2 transgenic mice. We have observed no obvious differences among BA.2, BA.4 and BA.5 isolates in growth ability or pathogenicity in rodent models, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 clinical isolates have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2.

Effectiveness of HEPA Filters at Removing Infectious SARS-CoV-2 from the Air.

Coronavirus disease 2019 (COVID-19) spreads by airborne transmission; therefore, the development and functional evaluation of air-cleaning technologies are essential for infection control. Air filtration using high-efficiency particulate air (HEPA) filters may be effective; however, no quantitative assessment of the effectiveness of these filters in the removal of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from the air has been reported. To evaluate the removal effect of HEPA filtration on airborne SARS-CoV-2, here, we disseminated infectious SARS-CoV-2 aerosols in a test chamber in a biosafety level 3 facility and filtered the air with a HEPA-filtered air cleaner in the chamber. The air cleaner with the HEPA filter continuously removed the infectious SARS-CoV-2 from the air in a running-time-dependent manner, and the virus capture ratios were 85.38%, 96.03%, and >99.97% at 1, 2, and 7.1 ventilation volumes, respectively. The air-cleaning performance of a HEPA filter coated with an antiviral agent consisting mainly of a monovalent copper compound was also evaluated, and the capture ratio was found to be comparable to that of the conventional HEPA filter. This study provides insights into the proper use and performance of HEPA-filtered air cleaners to prevent the spread of COVID-19. IMPORTANCE Air filtration simulation experiments quantitatively showed that an air cleaner equipped with a HEPA filter can continuously remove SARS-CoV-2 from the air. The capture ratios for SARS-CoV-2 in the air when the air cleaner was equipped with an antiviral-agent-coated HEPA filter were comparable to those with the conventional HEPA filter, and there was little effect on SARS-CoV-2 in the air that passed through the antiviral-reagent-coated HEPA filter.

Characterization and antiviral susceptibility of SARS-CoV-2 Omicron BA.2.

The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.

Therapeutic efficacy of monoclonal antibodies and antivirals against SARS-CoV-2 Omicron BA.1 in Syrian hamsters.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the major antigen stimulating the host's protective immune response. Here we assessed the efficacy of therapeutic monoclonal antibodies (mAbs) against Omicron variant (B.1.1.529) sublineage BA.1 variants in Syrian hamsters. Of the FDA-approved therapeutic mAbs tested (that is, REGN10987/REGN10933, COV2-2196/COV2-2130 and S309), only COV2-2196/COV2-2130 efficiently inhibited BA.1 replication in the lungs of hamsters, and this effect was diminished against a BA.1.1 variant possessing the S-R346K substitution. In addition, treatment of BA.1-infected hamsters with molnupiravir (a SARS-CoV-2 RNA-dependent RNA polymerase inhibitor) or S-217622 (a SARS-CoV-2 protease inhibitor) strongly reduced virus replication in the lungs. These findings suggest that the use of therapeutic mAbs in Omicron-infected patients should be carefully considered due to mutations that affect efficacy, and demonstrate that the antiviral compounds molnupiravir and S-217622 are effective against Omicron BA.1 variants.