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1.
Immunohorizons ; 6(4): 275-282, 2022 Apr 27.
Article in English | MEDLINE | ID: covidwho-1818325

ABSTRACT

Putative subcomponent vaccines of severe acute respiratory syndrome coronavirus spike protein and ARNAX (TLR3-specific adjuvant for priming dendritic cells) were examined and compared with spike protein + Alum in a mouse BALB/c model. Survival, body weight, virus-neutralizing Ab titer in the blood, and viral titer in the lung were evaluated for prognosis markers. The infiltration degrees of eosinophils in the lung were histopathologically monitored at 10 d postinfection. The results were: (1) adjuvant was essential in vaccines to achieve a complete recovery from infection, (2) ARNAX displayed optimal body weight recovery compared with Alum, (3) ARNAX was optimal for the amelioration of eosinophilic pneumonia, and (4) the eosinophil infiltration score was not associated with the neutralizing Ab titer in the blood or viral titer in the lung. Although the pathological link between the TLR3 vaccine and lung eosinophil infiltration remains unclear, severe acute respiratory syndrome-mediated eosinophilic pneumonia can be blocked by the prior induction of dendritic cell priming by ARNAX.


Subject(s)
Pulmonary Eosinophilia , SARS Virus , Viral Vaccines , Adjuvants, Immunologic/pharmacology , Animals , Body Weight , Dendritic Cells , Disease Models, Animal , Mice , Pulmonary Eosinophilia/prevention & control , Toll-Like Receptor 3 , Viral Vaccines/pharmacology
2.
Antiviral Res ; 201: 105297, 2022 May.
Article in English | MEDLINE | ID: covidwho-1814106

ABSTRACT

Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection.


Subject(s)
Antineoplastic Agents, Immunological , COVID-19 , Antibodies, Monoclonal , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing , Antibodies, Viral , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Combinations , Humans , Leukocytes, Mononuclear , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-318387

ABSTRACT

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.

4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-313038

ABSTRACT

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.

5.
Cell Rep Med ; 3(2): 100520, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1633496

ABSTRACT

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (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 investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is 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.


Subject(s)
Administration, Intranasal/methods , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19 Vaccines/administration & dosage , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Vaccination/methods , Animals , COVID-19/epidemiology , COVID-19/virology , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Coronavirus Envelope Proteins/immunology , Coronavirus M Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Disease Models, Animal , Female , Macaca fascicularis , Male , Pandemics/prevention & control , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Treatment Outcome , Vero Cells , Viral Load
6.
Sci Adv ; 8(1): eabh3827, 2022 Jan 07.
Article in English | MEDLINE | ID: covidwho-1612934

ABSTRACT

One safety concern during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development has been the vaccine-associated enhanced disease, which is characterized by eosinophilic immunopathology and T helper cell type 2 (TH2)­biased immune responses with insufficient neutralizing antibodies. In this study, we established a lethal animal model using BALB/c mice and a mouse-passaged isolate (QHmusX) from a European lineage of SARS-CoV-2. The QHmusX strain induced acute respiratory illness, associated with diffuse alveolar damage and pulmonary edema, in TH2-prone adult BALB/c mice, but not in young mice or TH1-prone C57BL/6 mice. We also showed that immunization of adult BALB/c mice with recombinant spike protein without appropriate adjuvant caused eosinophilic immunopathology with TH2-shifted immune response and insufficient neutralizing antibodies after QHmusX infection. This lethal mouse model is useful for evaluating vaccine-associated enhanced respiratory disease during SARS-CoV-2 infection and may provide new insights into the disease pathogenesis of SARS-CoV-2.

7.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-296101

ABSTRACT

Summary There are several recommendations for the use of personal protective equipment (PPE) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the selection of appropriate PPE for the current situation remains controversial. We measured serum antibody titers for SARS-CoV-2 in 10 participants who were engaged in the operation of charter flights for the evacuation of Japanese residents from Hubei Province. All participants wore PPE in accordance with Centers for Disease Control and Prevention guidelines. A total of 17 samples were tested, and all were seronegative. Hence, we conclude that the current PPE recommendation is effective to protect healthcare workers from SARS-CoV-2 infection.

8.
PLoS Pathog ; 17(7): e1009668, 2021 07.
Article in English | MEDLINE | ID: covidwho-1388961

ABSTRACT

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , COVID-19/veterinary , Macaca fascicularis/immunology , Macaca fascicularis/virology , Monkey Diseases/immunology , Monkey Diseases/virology , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/virology , Disease Models, Animal , Female , Humans , Kinetics , Lymphocyte Depletion/veterinary , Male , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , Virus Replication/immunology
9.
Immunity ; 54(10): 2385-2398.e10, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1370548

ABSTRACT

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


Subject(s)
Broadly Neutralizing Antibodies/immunology , Cross Reactions/immunology , SARS-CoV-2/immunology , Animals , Betacoronavirus/immunology , Binding Sites, Antibody , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/therapeutic use , COVID-19/prevention & control , COVID-19/therapy , COVID-19/virology , Cricetinae , Humans , Immunoglobulin Class Switching , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Mice , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
10.
Microbiol Immunol ; 64(1): 33-51, 2020 Jan.
Article in English | MEDLINE | ID: covidwho-1262996

ABSTRACT

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS-CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll-like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet-inactivated SARS-CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse-adapted SARS-CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP-adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro-inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist-adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia-associated coronaviruses.


Subject(s)
Adjuvants, Immunologic/pharmacology , Coronavirus Infections/prevention & control , Gold/chemistry , Immunoglobulin G/immunology , Lung/immunology , Metal Nanoparticles/chemistry , Severe Acute Respiratory Syndrome/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Analysis of Variance , Animals , Antibodies, Viral/immunology , Chlorocebus aethiops , Coronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cytokines/metabolism , Disease Models, Animal , Female , Immunization , Lung/pathology , Mice , Mice, Inbred BALB C , Recombinant Proteins/immunology , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/genetics , Toll-Like Receptors , Vaccination , Vaccines, Synthetic , Vero Cells , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Viral Vaccines/pharmacology , Viral Vaccines/therapeutic use
11.
Vaccines (Basel) ; 8(4)2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-902683

ABSTRACT

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 and causes severe and often fatal acute respiratory illness in humans. No approved prophylactic and therapeutic interventions are currently available. In this study, we have developed egg yolk antibodies (immunoglobulin Y (IgY)) specific for MERS-CoV spike protein (S1) in order to evaluate their neutralizing efficiency against MERS-CoV infection. S1-specific immunoglobulins were produced by injecting chickens with purified recombinant S1 protein of MERS-CoV at a high titer (5.7 mg/mL egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated that the IgY antibody specifically bound to the MERS-CoV S1 protein. Anti-S1 antibodies were also able to recognize MERS-COV inside cells, as demonstrated by an immunofluorescence assay. Plaque reduction and microneutralization assays showed the neutralization of MERS-COV in Vero cells by anti-S1 IgY antibodies and non-significantly reduced virus titers in the lungs of MERS-CoV-infected mice during early infection, with a nonsignificant decrease in weight loss. However, a statistically significant (p = 0.0196) quantitative reduction in viral antigen expression and marked reduction in inflammation were observed in lung tissue. Collectively, our data suggest that the anti-MERS-CoV S1 IgY could serve as a potential candidate for the passive treatment of MERS-CoV infection.

12.
J Infect Chemother ; 27(1): 120-122, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-753273

ABSTRACT

INTRODUCTION: Information on the effectiveness of personal protective equipment (PPE) for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among healthcare workers (HCWs), especially among HCWs with frequent contact with patients with SARS-CoV-2, is limited. METHODS: We conducted a prospective cohort study on 49 HCWs who worked in close contact with patients with SARS-CoV-2 infection. HCWs had blood samples taken every 2 weeks to test for SARS-CoV-2 antibodies using two different types of assay. RESULTS: Forty-nine participants (31 nurses, 15 doctors, 3 other workers) were enrolled. In total, 112 blood samples are obtained from participants. The median work days in 2 weeks was 9 (interquartile range (IQR): 5-10) days. In a single work day, 30 of the 49 participants (61.5%) had contact with patients with suspected or conformed SARS-CoV-2 at least 8 times, and approximately 60% of participants had more than 10 min of contact with a single patient. The median self-reported compliance to PPE was 90% (IQR: 80-100%). Seven participants tested positive for SARS-CoV-2 antibody using enzyme-linked immunosorbent assay (ELISA); however, none were seropositive for SARS-CoV-2 neutralizing antibody, so the positive ELISA results were assumed to be false-positive. CONCLUSIONS: The study provides evidence that appropriate PPE is sufficient to prevent infection amongHCWs. It is necessary to establish a system that provides a stable supply of PPE for HCWs to perform their duties.


Subject(s)
Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Health Personnel , Infectious Disease Transmission, Patient-to-Professional/prevention & control , Pandemics/prevention & control , Personal Protective Equipment , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Adult , Aged , Antibodies, Viral/blood , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged , Pneumonia, Viral/diagnosis , Prospective Studies , SARS-CoV-2 , Young Adult
13.
Proc Natl Acad Sci U S A ; 117(13): 7001-7003, 2020 03 31.
Article in English | MEDLINE | ID: covidwho-8451

ABSTRACT

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Serine Endopeptidases/metabolism , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Disease Outbreaks , Humans , Pandemics , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells , Virus Cultivation
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