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1.
Viruses ; 14(3)2022 03 06.
Article in English | MEDLINE | ID: covidwho-1765949

ABSTRACT

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.


Subject(s)
Adenoviruses, Human , Adenoviruses, Simian , Adenoviridae/genetics , Adenoviruses, Human/genetics , Adenoviruses, Simian/genetics , Animals , DNA , Genetic Vectors/genetics , Humans , Mammals
2.
Signal Transduct Target Ther ; 7(1): 61, 2022 02 25.
Article in English | MEDLINE | ID: covidwho-1758178

ABSTRACT

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Cross Protection , SARS-CoV-2/drug effects , Vaccination/methods , Vaccines, Inactivated/administration & dosage , Anal Canal/virology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/immunology , COVID-19/virology , Humans , Immunogenicity, Vaccine , Lung/virology , Macaca mulatta , Male , Nasal Cavity/virology , Pharynx/virology , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , T-Lymphocytes/immunology , T-Lymphocytes/virology , Viral Load/drug effects
3.
iScience ; 2022.
Article in English | EuropePMC | ID: covidwho-1755857

ABSTRACT

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is a complex with nutritional composition to nourish infants and protect them from different kinds of infectious diseases including COVID-19. Here, we identified lactoferrin (LF), mucin1 (MUC1) and α-lactalbumin (α-LA) from human breastmilk inhibit SARS-CoV-2 infection using a SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Additionally, LF and MUC1 inhibited multiple steps including viral attachment, entry and post-entry replication, while α-LA inhibited viral attachment and entry. Importantly, LF, MUC1 and α-LA possessed potent antiviral activities towards variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Taken together, our study provides evidence that human breastmilk components (LF, MUC1 and α-LA) are promising antiviral and potential therapeutic candidates warranting further development or treating COVID-19. Graphical

4.
Emerg Microbes Infect ; 11(1): 1058-1071, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1752040

ABSTRACT

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 µg or 50 µg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.


Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , Carrier Proteins , Humans , Immunogenicity, Vaccine , Mice , Mice, Inbred BALB C , Primates , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Subunit
5.
Nano today ; 2022.
Article in English | EuropePMC | ID: covidwho-1749472

ABSTRACT

While gold compound have been approved for Rheumatoid arthritis treatment as it well suppresses inflammatory cytokines of patients, no such treatment is currently available for COVID-19 treatment in vivo. We firstly disclose gold cluster yields better therapeutic outcome than Remdesivir in COVID-19 hamster treatments as it is armed with direct inhibition viral replication and intrinsic suppression inflammatory cytokines expression. Crystal data reveals that Au (I), released from gold cluster (GA), covalently binds thiolate of Cys145 of SARS-CoV-2 Mpro. GA directly decreases SARS-CoV-2 viral replication and intrinsically down-regulates NFκB pathway therefore significantly inhibiting expression of inflammatory cytokines in cells. The inflammatory cytokines in GA-treated COVID-19 transgenic mice are found to be significantly lower than that of control mice. When COVID-19 golden hamsters are treated by GA, the lung inflammatory cytokines levels are significantly lower than that of Remdesivir. The pathological results show that GA treatment significantly reduce lung inflammatory injuries when compared to that of Remdesivir-treated COVID-19 hamsters. Graphical

6.
Front Immunol ; 13: 827605, 2022.
Article in English | MEDLINE | ID: covidwho-1742217

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency of international concern, and an effective vaccine is urgently needed to control the pandemic. Envelope (E) and membrane (M) proteins are highly conserved structural proteins among SARS-CoV-2 and SARS-CoV and have been proposed as potential targets for the development of cross-protective vaccines. Here, synthetic DNA vaccines encoding SARS-CoV-2 E/M proteins (called p-SARS-CoV-2-E/M) were developed, and mice were immunised with three doses via intramuscular injection and electroporation. Significant cellular immune responses were elicited, whereas no robust humoral immunity was detected. In addition, novel H-2d-restricted T-cell epitopes were identified. Notably, although no drop in lung tissue virus titre was detected in DNA-vaccinated mice post-challenge with SARS-CoV-2, immunisation with either p-SARS-CoV-2-E or p-SARS-CoV-2-M provided minor protection and co-immunisation with p-SARS-CoV-2-E+M increased protection. Therefore, E/M proteins should be considered as vaccine candidates as they may be valuable in the optimisation of vaccination strategies against COVID-19.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , Coronavirus Envelope Proteins/genetics , Coronavirus M Proteins/genetics , SARS-CoV-2/physiology , T-Lymphocytes/immunology , Animals , Antibodies, Viral/blood , COVID-19 Vaccines/genetics , Female , Humans , Immunization , Mice , Mice, Inbred BALB C , Vaccines, DNA
7.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329581

ABSTRACT

Emerging in December 2019, coronavirus disease 2019 (COVID-19) eventually became a pandemic and has posed a tremendous threat to global public health. However, the origins of SARS-CoV-2, the causative agent of COVID-19, remain to be determined. It has reported that a certain number of the early case clusters had a contact history with Huanan Seafood Market. Therefore, surveillance of SARS-CoV-2 within the market is of vital importance. Herein, we presented the SARS-CoV-2 detection results of 1380 samples collected from the environment and the animals within the market in early 2020. By SARS-CoV-2-specific RT-qPCR, 73 environmental samples tested positive for SARS-CoV-2 and three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.980% to 99.993% with the human isolate HCoV/Wuhan/IVDC-HB-01. In contrast, no virus was detected in the animal swabs covering 18 species of animals in the market. The SARS-COV-2 nucleic acids in the positive environmental samples showed significant correlation of abundance of Homo sapiens with SARS-CoV-2. In summary, this study provided convincing evidence of the prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stage of COVID-19 outbreak.

8.
J Infect Dis ; 2021 Dec 27.
Article in English | MEDLINE | ID: covidwho-1704225

ABSTRACT

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic needs effective vaccines. METHODS: In a phase 2 randomized, double-blind, placebo-controlled trial, 500 adults aged 18-59 years or ≥60 years were randomized in 2:2:1 ratio to receive 3 doses of 5-µg or 10-µg of a SARS-CoV-2 inactivated vaccine, or placebo separated by 28 days. Adverse events (AEs) were recorded through Day 28 after each dosing. Live virus or pseudovirus neutralizing antibodies, and receptor binding domain (RBD-IgG) antibody were tested after the second and third doses. RESULTS: Two doses of the vaccine elicited geometric mean titers (GMTs) of 102-119, 170-176, and 1449-1617 for the three antibodies in younger adults. Pseudovirus neutralizing and RBD-IgG GMTs were similar between older and younger adults. The third dose slightly (<1.5 folds) increased GMTs. Seroconversion percentages were 94% or more after two doses, which were generally similar after three doses. The predominant AEs were injection-site pain. All the AEs were grade 1 or 2 in intensity. No serious AE was deemed related to study vaccination. CONCLUSIONS: Two doses of this vaccine induced robust immune response and had good safety profile. A third dose given 28 days after the second dose elicited limited boosting antibody response.

9.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323657

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved specific drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad- spectrum antiviral drugs. Here, based on the highly similar structure of SARS- CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under- clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro . Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These findings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan- coronavirus antiviral agents.Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.

10.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-312727

ABSTRACT

The rapid expansion of COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantages of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates has entered clinical trials with a two-dose immunization regimen. However, the waning antibodies response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concern about a short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form which encoded SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular response, and conferred a near-complete protection against wild SARS-CoV-2 infection in lungs of hACE2 transgenic mice. Noticeably, high levels of neutralizing antibodies response induced by mRNA-RBD vaccination could maintain for at least 6.5 months and conferred a long-term remarkable protection for hACE2 transgenic mice against SARS-CoV-2 infection in sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.

12.
Am J Physiol Lung Cell Mol Physiol ; 321(5): L958-L959, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1526552

Subject(s)
COVID-19 , China , Humans , SARS-CoV-2
13.
ACS Med Chem Lett ; 12(11): 1838-1844, 2021 Nov 11.
Article in English | MEDLINE | ID: covidwho-1507014

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has stimulated the search for effective drugs for its prevention and treatment. Natural products are an important source for new drug discovery. Here, we report that, NK007(S,R), a tylophorine malate, displays high antiviral activity against SARS-CoV-2 with an EC50 0.03 µM in vitro, which is substantially lower than that of remdesivir (EC50: 0.8 µM in vitro), the only authorized drug to date. The histopathological research revealed that NK007(S,R) (5 mg/kg/dose) displayed a protection effect in lung injury induced by SARS-CoV-2, which is better than remdesivir (25 mg/kg/dose). We also prepared two nanosized preparations of NK007(S,R), which also showed good efficacy (EC50: NP-NK007, 0.007 µM in vitro; LP-NK007, 0.014 µM in vitro). Our findings suggest that tylophora alkaloids, isolated from the traditional Chinese medicine Cynanchum komarovii AL, offer a new skeleton for the development of anticoronavirus drug candidate.

14.
Infect Dis Ther ; 11(1): 165-174, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1482329

ABSTRACT

INTRODUCTION: Since the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19. METHODS: We conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1-3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children's Hospital of Fudan University during 2010-2020 were analyzed. RESULTS: Of the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September. CONCLUSIONS: These eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

18.
China CDC Wkly ; 2(25): 453-457, 2020 Jun 19.
Article in English | MEDLINE | ID: covidwho-1449640

ABSTRACT

WHAT IS ALREADY KNOWN ON THIS TOPIC?: A novel human coronavirus, known as SARS-CoV-2 or 2019-nCoV, is the causative agent of the coronavirus disease 2019 (COVID-19). We have released the primers and probes of real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the laboratory detection of COVID-19 infection. WHAT IS ADDED BY THIS REPORT?: Here we provide detailed technical data and evaluate the performance of three novel rRT-PCR assays targeting the ORF1ab, N, and E genes for detection of COVID-19 infection. The application of rRT-PCR assays among four types of specimens (alveolar lavage, sputum, throat swabs, and stool) from patients with COVID-19 indicated that the mean viral loads detected in sputum were higher than other specimens. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: These rRT-PCR assays reported here could be used for laboratory diagnosis of COVID-19 infection with high sensitivity, specificity, and applicability. Sputum rather than throat swabs and stool should be a priority for specimen collection for laboratory detection of COVID-19.

19.
China CDC Wkly ; 2(25): 447-452, 2020 Jun 19.
Article in English | MEDLINE | ID: covidwho-1449635

ABSTRACT

What is already known on this topic? Coronavirus disease 2019 (COVID-19), a disease caused by a novel human coronavirus named the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19 virus, was reported in December 2019. Complete genomes of the COVID-19 virus from clinical samples using next generation sequencing (NGS) have been reported. What is added by this report? Here we provide the technical data for sequencing complete genome of COVID-19 virus from clinical samples using the Sanger method. Two complete COVID-19 virus genome sequences (named WH19004-S and GX0002) were obtained from clinical samples of COVID-19 patients, and two single nucleotide polymorphisms (SNPs) in ORF7a (T/C, nt 27,493) and ORF8 (T/C, nt 28,253) of WH19004-S were identified by Sanger sequencing. What are the implications for public health practice? The COVID-19 virus genome sequencing by Sanger method reported here could be used to generate data of high enough quality without requirement for expensive NGS equipment, which support sequencing complete genomes from clinical samples and monitoring of viral genetic variations of COVID-19 infections.

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