Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies (preprint)

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

Estimating serum cross-neutralizing responses to SARS-CoV-2 Omicron sub-lineages elicited by pre-Omicron or Omicron breakthrough infection with exposure interval compensation modeling (preprint)

Understanding the differences in serum cross-neutralizing responses against SARS-CoV-2 variants, including Omicron sub-lineages BA.5, BA.2.75, and BQ.1.1, elicited by exposure to distinct antigens is essential for developing COVID-19 booster vaccines with enhanced cross-protection against antigenically distinct variants. However, fairly comparing the impact of breakthrough infection on serum neutralizing responses to several variants with distinct epidemic timing is challenging because responses after breakthrough infection are affected by the exposure interval between vaccination and infection. We assessed serum cross-neutralizing responses to SARS-CoV-2 variants, including Omicron sub-lineages, in individuals with breakthrough infections before or during the Omicron BA.1 epidemic. To understand the differences in serum cross-neutralizing responses after pre-Omicron or Omicron breakthrough infection, we used Bayesian hierarchical modeling to correct the cross-neutralizing responses for the exposure interval between vaccination and breakthrough infection. The exposure interval required to generate saturated cross-neutralizing potency against each variant differed by variant, with variants more antigenically distant from the ancestral strain requiring a longer interval. Additionally, Omicron breakthrough infection was estimated to have higher impact than booster vaccination and pre-Omicron breakthrough infection on inducing serum neutralizing responses to the ancestral strain and Omicron sub-lineages. However, the breadth of cross-neutralizing responses to Omicron sub-lineages, including BQ.1.1, after Omicron or pre-Omicron breakthrough infection with the ideal exposure interval were estimated to be comparable. Our results highlight the importance of optimizing the interval between vaccine doses for maximizing the breadth of cross-neutralizing activity elicited by booster vaccines with or without Omicron antigen.

Heterologous booster immunization with SARS-CoV-2 spike protein after mRNA vaccine elicits durable and broad antibody responses (preprint)

Although mRNA vaccines are more immunogenic than other vaccine modalities in primary series vaccination, their immunogenicity has not been well compared to different vaccine modalities in additional boosters. Here the longitudinal analysis reveals more sustained RBD-binding IgG titers and RBD-ACE2 binding inhibitory activities with the breadth to antigenically distinct Beta and Omicron BA.1 variants by the S-268019-b spike protein booster vaccination compared to BNT162b2 mRNA homologous booster on mRNA vaccinees. The differences in the durability and breadth of plasma antibodies between BNT162b2 and S-268019-b groups are pronounced in those without systemic adverse events and were associated with different trends in the number and breadth of memory B cells. High-dimensional immune profiling identifies early CD16 + natural killer cell dynamics with CCR3 upregulation, as one of the correlates for the distinct antibody responses by the S-268019-b booster. Our results illustrate the combinational effects of heterologous booster on the immune dynamics and the durability and breadth of recalled antibody responses against emerging virus variants.

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models. (preprint)

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 COVID-19-convalescent patients, and identified antibodies that exhibited 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, and 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.

Viral load and timing of infection define neutralization diversity to SARS-CoV-2 infection (preprint)

AbstractImmunity to SARS-CoV-2 in COVID-19 cases has diversified due to complex combinations of exposure to vaccination and infection. Elucidating the drivers for upgrading neutralizing activity to SARS-CoV-2 in COVID-19 cases with pre-existing immunity will aid in understanding immunity to SARS-CoV-2 and improving COVID-19 booster vaccines with enhanced cross-protection against antigenically distinct variants. This study revealed that the magnitude and breadth of neutralization responses to SARS-CoV-2 infection in breakthrough infections are determined by upper respiratory viral load and vaccination-infection time interval, but not by the lineage of infecting viruses. Notably, the time interval, but not the viral load, may play a critical role in expanding the breadth of neutralization to SARS-CoV-2. This illustrates the importance of dosing interval optimization in addition to antigen design in the development of variant-proof booster vaccines. One-Sentence SummaryViral load and infection timing define the magnitude and breadth of SARS-CoV-2 neutralization after breakthrough infection.

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2 (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into the impact of variant emergence on choosing optimal drug treatment.

Safety and immunogenicity of Pfizer/BioNTech SARS-CoV-2 mRNA third booster vaccine against SARS-CoV-2 Omicron variant in Japanese healthcare workers (preprint)

Background: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in Japan in November 2021. This variant contains up to 36 mutations in the spike protein, the target of neutralizing antibodies, and can escape vaccine-induced immunity. The third booster vaccination campaign began with healthcare workers and high-risk groups. The safety and immunogenicity of third booster vaccination against Omicrons remain unknown. Methods: In total, 272 healthcare workers were evaluated for their long-term safety and immunogenicity. Here, we established vaccine panels to evaluate the safety and immunogenicity against variants of concern (VOCs), including the Omicron variant, using a live virus microneutralization assay. Findings: Two-dose vaccination induced robust anti-spike antibodies and neutralization titers (NTs) against the ancestral strain WK-521, whereas NTs in VOCs were significantly decreased. Within 93-247 days of the second vaccine dose, NTs against Omicron were completely abolished in up to 80% of individuals among the vaccine panels. The third booster vaccination induced a robust increase in anti-spike antibodies and NTs against the WK-521, Delta, and Omicron variants. The breadth of humoral immunity and cross-reactivity with Omicron increased. The cytokine signature and adverse event rate remained unchanged after three dose vaccination. Conclusions: The third vaccination dose is safe and effective against Omicron infection.

Vaccination-infection interval determines cross-neutralization potency to SARS-CoV-2 Omicron after breakthrough infection by other variants (preprint)

Background The immune profile against SARS-CoV-2 has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by the Omicron in individuals with various immune histories. Methods The neutralization susceptibility of the variants including the Omicron and their ancestor was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections by the Alpha/Delta with multiple time intervals following vaccination. Findings The Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against the Omicron were induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions Immune histories with breakthrough infections can overcome the resistance to infection by the Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against the Omicron and future variants. Funding This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).

Two doses of mRNA vaccine elicit cross-neutralizing memory B-cells against SARS-CoV-2 Omicron variant (preprint)

SARS-CoV-2 Beta and Omicron variants have multiple mutations in the receptor-binding domain (RBD) allowing antibody evasion. Despite the resistance to circulating antibodies in those who received two doses of mRNA vaccine, the third dose prominently recalls cross-neutralizing antibodies with expanded breadth to these variants. Herein, we longitudinally profiled the cellular composition of persistent memory B-cell subsets and their antibody reactivity against these variants following the second vaccine dose. The vaccination elicited a memory B-cell subset with resting phenotype that dominated the other subsets at 4.9 months. Notably, most of the resting memory subset retained the ability to bind the Beta variant, and the memory-derived antibodies cross-neutralized the Beta and Omicron variants at frequencies of 59% and 29%, respectively. The preservation of cross-neutralizing antibody repertoires in the durable memory B-cell subset likely contributes to the prominent recall of cross-neutralizing antibodies following the third dose of the vaccine. One Sentence Summary Fully vaccinated individuals preserve cross-neutralizing memory B-cells against the SARS-CoV-2 Omicron variant.

Distinct immune cell dynamics mark adverse events and antibody responses of SARS-CoV-2 mRNA vaccine (preprint)

Pfizer/BioNTec BNT162b2 mRNA vaccine robustly elicits neutralizing antibodies against SARS-CoV-2 in clinical trials and real-world settings. However, booster vaccinations are frequently associated with self-limited adverse events. Here, by applying a high-dimensional immune profiling approach to peripheral blood, we linked early vaccine-induced immune dynamics with adverse events and neutralizing antibody responses. The dynamics of two dendritic cell subsets (DC3s and AS-DCs) were identified as the specific correlates for adverse events; the combination of these cell dynamics stratified the vaccinees with severe reactogenicity, while the stratification did not affect the neutralizing antibody titers. Furthermore, the NKT-like cell dynamics that correlated with adverse events and antibody titers were accounted for distinct magnitudes of both events by sex and age. The identified immune correlates for adverse events and antibody responses may pave the way for a rational vaccine strategy for reducing the reactogenicity of mRNA vaccines without compromising the immunogenicity.

Neutralizing Antibody-Independent SARS-CoV-2 Control Correlated with Intranasal Vaccine-Induced CD8 + T-Cell Responses (preprint)

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigated the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Nine vaccinated macaques exhibited significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs was significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment.Funding: This work was supported by Japan Agency for Medical Research and Development (AMED [JP19fk0108104 to A.K.-T. and JP20nk0101601, JP20jm0110012, JP21fk0410035, JP21fk0108125, and JP21jk0210002 to T.M.]) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (JSPS Grants-in-Aid for Scientific Research [21H02745 to T.M.]).Declaration of Interests: H.I., K.K., R.S., and T.M are the inventors on Patent Cooperation Treaty (PCT) application for SeV-NME vaccine. Authors have no other conflicts of interest to declare.Ethics Approval Statement: Approval by the Committee on the Ethics of Animal Experiments in NIID (permission number: 520001) under the guidelines for animal experiments in accordance with the Guidelines for Proper Conduct of Animal Experiments established by the Science Council of Japan.

Viral and Immunological Characteristics of COVID-19 Vaccine Breakthrough Infections (preprint)

Since little is known about viral and host characteristics of breakthrough infections after COVID-19 vaccination, a nationwide investigation of breakthrough cases was initiated in Japan. 130 cases (90%+ received mRNA vaccines) were reported with respiratory specimens in 117 cases and sera in 68 cases. A subset of cases shed infectious virus regardless of symptom presence or viral lineages. Viral lineages for breakthrough infections matched both temporally and spatially with the circulating lineages in Japan with no novel mutations in spike receptor binding domain that may have escaped from vaccine-induced immunity were found. Anti-spike/neutralizing antibodies of breakthrough infections in the acute phase owing to vaccine-induced immunity were significantly higher than those from unvaccinated convalescent individuals but were comparable to vaccinated uninfected individuals, and followed by boosting in the convalescent phase. Symptomatic cases had low anti-spike/neutralizing antibodies in the acute phase with robust boosting in the convalescent phase, suggesting the presence of serological correlate for symptom development in COVID-19 vaccine breakthrough infections.

Antibody responses to BNT162b2 vaccination in Japan: Monitoring vaccine efficacy by measuring IgG antibodies against the receptor binding domain of SARS-CoV-2 (preprint)

BACKGROUNDTo fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), mass vaccination has begun in many countries. To investigate the usefulness of a serological assay to predict vaccine efficacy, we analyzed the levels of IgG, IgM, and IgA against the receptor binding domain (RBD) of SARS-CoV-2 in the sera from BNT162b2 vaccinated individuals in Japan. METHODSThis study included 219 individuals who received two doses of BNT162b2. The levels of IgG, IgM, and IgA against RBD were measured by enzyme-linked immunosorbent assay before and after the first and second vaccination, respectively. The relationship between antibody levels and several factors including age, gender, and hypertension were analyzed. Virus-neutralizing activity in sera was measured to determine the correlation with the levels of antibodies. A chemiluminescent enzyme immunoassay (CLEIA) method to measure IgG against RBD was developed and validated for the clinical setting. RESULTSThe levels of all antibody isotypes were increased after vaccination. Among them, RBD-IgG was dramatically increased after the second vaccination. The IgG levels in females were significantly higher than in males. There was a negative correlation between age and IgG levels in males. The IgG levels significantly correlated with the neutralizing activity. The CLEIA assay measuring IgG against RBD showed a reliable performance and a high correlation with neutralizing activity. CONCLUSIONSMonitoring of IgG against RBD is a powerful tool to predict the efficacy of SARS-CoV-2 vaccination and provides useful information in considering a personalized vaccination strategy for COVID-19.

Broad Neutralization Activity of SARS-CoV-2 Antibody is Achieved by Coordinated Recognition of Virus Vulnerable Site (preprint)

Potently neutralizing SARS-CoV-2 antibodies often target the receptor binding site (RBS) of spike protein but the variability of RBS epitopes hampers broad neutralization of different clades of coronaviruses and emerging drifted viruses. Here, we identified a human RBS antibody that potently neutralizes SARS-CoV and SARS-CoV-2 variants that belong to clade 1 SARS-related coronavirus. X-ray crystallography revealed coordinated recognition by the heavy chain to conserved sites and the light chain to RBS, allowing for the mimicry of ACE2 binding mode. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, and the activity was further enhanced by IgG3 switching. Eventually, the coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Furthermore, therapeutic treatment in a hamster model provided protection at low dosage. The structural basis for broadly neutralizing activity informs the design of broad spectrum of therapeutics and vaccines.Funding: This work was supported by Japan Agency for Medical Research and Development grant JP19fk0108111 (TH, YT), JP20fk0108298 (TK, TH, KM, YT), JP20am0101093 (KM), JP20ae0101047 (KM), JP20fk0108251 (HS), and JP20am0101124 (YK), by Ministry of Education, Culture, Sports, Science and Technology grant JPMXS0420100119 (KM) and 20H05773 (TH), by The Naito Foundation (TH), and by Joint Usage/Research Center program of Institute for Frontier Life and Medical Sciences, Kyoto University (KM).Conflict of Interest: AS is an employee of Shionogi & Co., Ltd. MO is a CEO, employee, and shareholder of Trans Chromosomics, Inc. These authors acknowledge a potential conflict of interest and attest that the work contained in this report is free of any bias that might be associated with the commercial goals of the company. TO, YA, MO, TH, KM, and YT declare that an intellectual property application has been filed using the data presented in this paper. The other authors declare that they have no competing interests.Ethical Approval: Animal procedures were approved by the Animal Ethics Committee of the National Institute of Infectious Diseases, Japan, and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. In vitro escape mutation screening experiments for SARSCoV-2 were performed at the Biosafety Level-3 facility of the Research Center for ZoonosisControl, Hokkaido University, and the National Institute of Infectious Diseases following the institutional guidelines.

Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients (preprint)

T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.

Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients (preprint)

Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide (preprint)

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. STATEMENT OF SIGNIFICANCESARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed "fusion peptide" (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

Anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) potency of Mefloquine as an entry inhibitor in vitro (preprint)

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 M, IC90 = 2.31 M, and IC99 = 4.39 M in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

Myeloid cell dynamics correlate with clinical outcomes of severe coronavirus disease 2019 (preprint)

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19); however, it remains unclear whether myeloid cells are beneficial or detrimental to the clinical outcome. Here, we tracked cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis by flow cytometric analysis of blood samples from COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, rather than other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Notably, this subset was selectively expanded in survivors of severe cases and diminished during recovery. Analysis of plasma cytokines/chemokines revealed that interleukin-8 increased prior to PMN-MDSC expansion in survivors and returned to basal levels during the recovery phase. In contrast, interleukin-6 and interferon-γ-induced protein 10 were abundantly induced in non-survivors, suggesting possible downstream targets for the immunosuppressive effects of the MDSC subset. Our data indicate that increased cellularity of PMN-MDSCs might be beneficial for the clinical outcome and could be useful as a possible predictor of prognosis in cases of severe COVID-19.