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1.
iScience ; 25(11): 105412, 2022 Nov 18.
Article in English | MEDLINE | ID: covidwho-2083114

ABSTRACT

Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37°C-39°C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.

2.
Immunol Med ; : 1-14, 2022 Aug 24.
Article in English | MEDLINE | ID: covidwho-1997042

ABSTRACT

There is an urgent need to stop the coronavirus disease 2019 (COVID-19) pandemic through the development of efficient and safe vaccination methods. Over the short term, plasmid DNA vaccines can be developed as they are molecularly stable, thus facilitating easy transport and storage. pVAX1-SARS-CoV2-co was designed for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) S protein. The antibodies produced led to immunoreactions against the S protein, an anti-receptor-binding-domain, and a neutralizing action of the pVAX1-SARS-CoV2-co, as previously confirmed. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine a pyro-drive jet injector (PJI) was used. An intradermally adjusted PJI demonstrated that the pVAX1-SARS-CoV2-co vaccine injection caused a high production of anti-S protein antibodies, triggered immunoreactions, and neutralized the actions against SARS-CoV-2. A high-dose pVAX1-SARS-CoV2-co intradermal injection using PJI did not cause any serious disorders in the rat model. A viral challenge confirmed that intradermally immunized mice were potently protected from COVID-19. A pVAX1-SARS-CoV2-co intradermal injection using PJI is a safe and promising vaccination method for overcoming the COVID-19 pandemic.

3.
Int J Infect Dis ; 122: 123-129, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1889491

ABSTRACT

BACKGROUND: From late March through April 2021, we experienced a cluster of patients with COVID-19, named "Cluster K", with rapid severe illness compared with those who were infected before. METHODS: Patients with COVID-19 who were enrolled in this study were divided into two groups: 66 patients from November 2020 to March 2021 (group A) and 37 patients whose infection links were traced from Cluster K (group B). The primary outcome was mortality rate, and the secondary outcome was maximal oxygen flow rate as the severity of the disease. Viral genome sequences were compared between the two groups. RESULTS: Mortality rates were 6.1% in group A and 16.2% in group B (odds ratio: 2.97, 95% confidence interval: 0.65-15.38). The patients in group B required high oxygen flow rate (O2 ≥10 l/min) in the earlier clinical course (P = 0.029). Viral genome sequences revealed five amino acid mutations; of these, four were found on three nonstructural proteins (NSPs): one in nsp3 and nsp15, two in nsp6 (one of them is near the potential sites under positive selective pressure). Another one was on the S protein. CONCLUSION: This study suggests that mutations in NSPs, especially nsp6, are associated with adverse clinical outcome in patients with COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , Humans , Mutation , Oxygen , SARS-CoV-2/genetics
4.
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases ; 2022.
Article in English | EuropePMC | ID: covidwho-1837401

ABSTRACT

Background . From late March through April 2021, we experienced a cluster of COVID-19 patients, named “Cluster K”, with rapid severe illness compared to those who were infected before. Method . COVID-19 patients enrolled in this study were divided into two groups: 66 patients from November 2020 to March 2021(group A), 37 patients whose infection links were traced from Cluster K (group B). The primary outcome was mortality rate, and the secondary outcome was maximal oxygen flow rate as severity of the disease. Viral genome sequences were compared between two groups. Results . Mortality rates were 6.1% in group A and 16.2% in group B (OR: 2.97, 95%CI: 0.65-15.38). The patients in group B required high oxygen flow rate (O2 ≥10 L/min) in earlier clinical course (p=0.029). Viral genome sequences revealed five amino acid mutations. Of these, four were found on three non-structural proteins (NSPs): one in nsp3 and 15, two in nsp6 (one of them is near the potential sites under positive selective pressure). Other one was on S protein. Conclusion . This study suggests that mutations in NSPs, especially nsp6, are associated with adverse clinical outcome in COVID-19 patients.

5.
J Pharmacol Sci ; 149(3): 81-84, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1796436

ABSTRACT

Ciclesonide (Cic) is approved as an inhalant for asthma and was clinically tested as a candidate therapy for coronavirus disease 2019 (COVID-19). Its active metabolite Cic2 was recently reported to suppress genomic RNA replication of severe acute respiratory syndrome coronavirus 2. In this study, we designed and synthesized a set of ciclesonide-acetal (Cic-acetal) derivatives. Among designated compounds, some Cic-acetal derivatives with a linear alkyl chain exhibited strong viral copy-number reduction activities compared with Cic2. These compounds might serve as lead compounds for developing novel anti-COVID-19 agents.


Subject(s)
Antiviral Agents , COVID-19 , Acetals/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Pregnenediones , RNA, Viral/genetics , RNA, Viral/pharmacology , SARS-CoV-2 , Virus Replication/genetics
6.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329088

ABSTRACT

Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

7.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-317457

ABSTRACT

Many variants that naturally acquire multiple mutations have emerged during the current SARS-CoV-2 pandemic, which is devastating societies worldwide. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here, we demonstrate that two recently emerging mutations in the receptor - binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce the affinity toward the viral receptor ACE2;notably, the L452R mutation increases spike stability and viral infectivity and potentially promotes viral replication. Our data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.Funding: This study was supported in part by AMED Research Program on Emerging and Re-emerging Infectious Diseases 20fk0108163 (to A.S.), 20fk0108146 (to K.S.), 19fk0108171 (to S.N. and K.S.), 20fk0108270 (to K.S.) and 20fk0108413 (to T.I., S.N. and K.S.);AMED Research Program on HIV/AIDS 20fk0410019 (to T.U. and K.S.), 20fk0410014 (to K.S.) and 21fk0410039 (to K.S.);AMED Japan Program for Infectious Diseases Research and Infrastructure 20wm0325009 (to A.S.);JST J RAPID JPMJJR2007 (to K.S.);JST SICORP (e-ASIA) JPMJSC20U1 (to K.S.);JSTCREST JPMJCR20H6 (to S.N) and JPMJCR20H4 (to K.S);JSPS KAKENHI Grant-in-Aid for Scientific Research B 18H02662 (to K.S.) and 21H02737 (to K.S.);JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas 16H06429 (to S.N. and K.S.), 16K21723 (to S.N. and K.S.), 17H05823 (to S.N.), 17H05813 (to K.S.), 19H04843 (to S.N.) and 19H04826 (to K.S.);JSPS Fund for the Promotion of Joint International Research (Fostering Joint International Research) 18KK0447 (to K.S.);JSPS Core-to-Core Program JPJSCCB20190009 (to T.U.) andJPJSCCA20190008 (A. Advanced Research Networks) (to K.S.);JSPS Research Fellow DC1 19J20488 (to I.K.);JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to T.I.);ONO Medical Research Foundation (to K.S.);Ichiro Kanehara Foundation (to K.S.);Lotte Foundation (to K.S.);Mochida Memorial Foundation for Medical and Pharmaceutical Research (to K.S.);Daiichi Sankyo Foundation of Life Science (to K.S.);Sumitomo Foundation (to K.S.);Uehara Foundation (to K.S.);Takeda Science Foundation (to C.M., T.I. and K.S.);The Tokyo Biochemical Research Foundation (to K.S.);Mitsubishi Foundation (to T.I.);Shin Nihon Foundation of Advanced Medical Research (to T.I.);An intramural grant from Kumamoto University COVID-19 Research Projects (AMABIE) (to C.M., T.I. and T.U.);Kumamoto University International Collaborative Research Grants (to T.U.);Intercontinental Research and Educational Platform Aiming for Eradication of HIV/AIDS (to T.I. and T.U.);2020 Tokai University School of Medicine Research Aid (to S.N.);and Joint Usage/Research Center program of Institute for Frontier Life and Medical Sciences, Kyoto University (to K.S.). T.S.T and I.N. are the recipients of the doctoral course scholarship from Japanese Government.Conflict of Interest: The authors declare that no competing interests exist.Ethical Approval: All protocols involving human subjects recruited at Kyushu University Hospital, Japan, National Hospital Organization Kyushu Medical Center, Japan, and Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Japan, were reviewed and approved by the Ethics Committee for Epidemiological andGeneral Research at the Faculty of Life Science, Kumamoto University (approval numbers 2066 and 461). All human subjects provided written informed consent.

8.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-295000

ABSTRACT

Among a cluster of COVID-19 cases from the end of March through April 2021 in Asahikawa, we experienced the cases in which patients manifested severe clinical symptoms compared to patients who were infected before that. A hundred three patients (age range: 65 to 89 years old) enrolled in this study were divided into two groups, group A: the patients infected from November 2020 to March 2021, and group B: the patients in this cluster population. The mortality rates were 6.1% in group A and 16.2% in group B (OR: 2.97, 95%CI: 0.65-15.38). For the severity of disease, the patients in group B required higher oxygen flow rate in early course of admission (mild;p=0.892, moderate;p=0.117, severe;p=0.029). Whole viral genome sequences revealed five non-synonymous mutations by comparison of the isolates with each group. Of these, four were on non-structural proteins (NSPs) including nsp3, 6 and 15, and one was on S protein located near the C-terminus, suggesting that the mutations on NSPs could be responsible for adverse clinical outcomes in COVID-19 patients.

9.
Cell Host Microbe ; 29(7): 1124-1136.e11, 2021 07 14.
Article in English | MEDLINE | ID: covidwho-1272337

ABSTRACT

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.


Subject(s)
COVID-19/virology , Immunity, Cellular , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , COVID-19/epidemiology , Genome, Viral , Humans , Mutation , Phylogeny , Protein Binding , Viral Proteins/genetics , Virus Replication
10.
Cell ; 184(13): 3452-3466.e18, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1240207

ABSTRACT

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , COVID-19/immunology , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Protein Binding/immunology , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
11.
Cell Rep ; 35(3): 109014, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1163485

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.


Subject(s)
COVID-19/genetics , Reverse Genetics , SARS-CoV-2/genetics , Animals , Cricetinae , HEK293 Cells , Humans
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