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1.
Lancet ; 398(10296): 213-222, 2021 07 17.
Article in English | MEDLINE | ID: covidwho-1598580

ABSTRACT

BACKGROUND: CoronaVac, an inactivated whole-virion SARS-CoV-2 vaccine, has been shown to be well tolerated with a good safety profile in individuals aged 18 years and older in phase 1/2 trials, and provided a good humoral response against SARS-CoV-2. We present the interim efficacy and safety results of a phase 3 clinical trial of CoronaVac in Turkey. METHODS: This was a double-blind, randomised, placebo-controlled phase 3 trial. Volunteers aged 18-59 years with no history of COVID-19 and with negative PCR and antibody test results for SARS-CoV-2 were enrolled at 24 centres in Turkey. Exclusion criteria included (but were not limited to) immunosuppressive therapy (including steroids) within the past 6 months, bleeding disorders, asplenia, and receipt of any blood products or immunoglobulins within the past 3 months. The K1 cohort consisted of health-care workers (randomised in a 1:1 ratio), and individuals other than health-care workers were also recruited into the K2 cohort (randomised in a 2:1 ratio) using an interactive web response system. The study vaccine was 3 µg inactivated SARS-CoV-2 virion adsorbed to aluminium hydroxide in a 0·5 mL aqueous suspension. Participants received either vaccine or placebo (consisting of all vaccine components except inactivated virus) intramuscularly on days 0 and 14. The primary efficacy outcome was the prevention of PCR-confirmed symptomatic COVID-19 at least 14 days after the second dose in the per protocol population. Safety analyses were done in the intention-to-treat population. This study is registered with ClinicalTrials.gov (NCT04582344) and is active but no longer recruiting. FINDINGS: Among 11 303 volunteers screened between Sept 14, 2020, and Jan 5, 2021, 10 218 were randomly allocated. After exclusion of four participants from the vaccine group because of protocol deviations, the intention-to-treat group consisted of 10 214 participants (6646 [65·1%] in the vaccine group and 3568 [34·9%] in the placebo group) and the per protocol group consisted of 10 029 participants (6559 [65·4%] and 3470 [34·6%]) who received two doses of vaccine or placebo. During a median follow-up period of 43 days (IQR 36-48), nine cases of PCR-confirmed symptomatic COVID-19 were reported in the vaccine group (31·7 cases [14·6-59·3] per 1000 person-years) and 32 cases were reported in the placebo group (192·3 cases [135·7-261·1] per 1000 person-years) 14 days or more after the second dose, yielding a vaccine efficacy of 83·5% (95% CI 65·4-92·1; p<0·0001). The frequencies of any adverse events were 1259 (18·9%) in the vaccine group and 603 (16·9%) in the placebo group (p=0·0108) with no fatalities or grade 4 adverse events. The most common systemic adverse event was fatigue (546 [8·2%] participants in the vaccine group and 248 [7·0%] the placebo group, p=0·0228). Injection-site pain was the most frequent local adverse event (157 [2·4%] in the vaccine group and 40 [1·1%] in the placebo group, p<0·0001). INTERPRETATION: CoronaVac has high efficacy against PCR-confirmed symptomatic COVID-19 with a good safety and tolerability profile. FUNDING: Turkish Health Institutes Association.


Subject(s)
Antibodies, Neutralizing , COVID-19 Vaccines/therapeutic use , COVID-19/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/prevention & control , Double-Blind Method , Health Personnel/statistics & numerical data , Humans , Male , Middle Aged , Turkey , Vaccination , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Virion/immunology
2.
Bull Exp Biol Med ; 171(2): 230-233, 2021 May.
Article in English | MEDLINE | ID: covidwho-1525548

ABSTRACT

The presence of IgG and IgM antibodies in the venous blood of 76 patients with confirmed COVID-19 infection was determined by ELISA using Russian test systems. Different levels of IgM antibodies to N-protein and receptor binding domain of the Spike protein (RBD) were revealed. The dynamics of IgG antibodies to the whole virion antigen and recombinant antigens showed high values on weeks 4-5 of the disease. The level of IgG antibodies to Nprotein remained low throughout the observation period. The characteristic dynamics of IgG measured using test systems with sorbed whole virion or recombinant spike proteins reflects the duration of the disease.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Spike Glycoprotein, Coronavirus/immunology , Antigens, Viral/genetics , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Immunity, Humoral , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Time Factors , Virion/genetics , Virion/immunology
3.
Virus Res ; 305: 198555, 2021 11.
Article in English | MEDLINE | ID: covidwho-1412516

ABSTRACT

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Vaccines/chemistry , Propiolactone/pharmacology , SARS-CoV-2/drug effects , Virion/drug effects , Virus Inactivation/drug effects , Animals , Antigens, Viral/chemistry , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Flocculation/drug effects , Humans , Immune Sera/chemistry , RNA, Viral/chemistry , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Vaccines, Inactivated , Vero Cells , Virion/chemistry , Virion/immunology
4.
Virol J ; 18(1): 1, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-1388776

ABSTRACT

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.


Subject(s)
COVID-19/immunology , Leukemia/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , HEK293 Cells , Humans , Immunoglobulin G/blood , Mice
6.
Int J Mol Sci ; 22(16)2021 Aug 23.
Article in English | MEDLINE | ID: covidwho-1367852

ABSTRACT

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.


Subject(s)
Antibodies, Viral/metabolism , COVID-19/immunology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/pathogenicity , Viral Matrix Proteins/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , Cell Line , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/immunology , Coronavirus Envelope Proteins/ultrastructure , Cricetinae , Humans , Microscopy, Electron, Transmission , Mutation , Neutralization Tests , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Matrix Proteins/ultrastructure , Virion/genetics , Virion/immunology , Virion/metabolism , Virion/ultrastructure
7.
Front Immunol ; 12: 669103, 2021.
Article in English | MEDLINE | ID: covidwho-1348487

ABSTRACT

Targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), especially severe cases, are currently lacking. As macrophages have unique effector functions as a first-line defense against invading pathogens, we genetically armed human macrophages with chimeric antigen receptors (CARs) to reprogram their phagocytic activity against SARS-CoV-2. After investigation of CAR constructs with different intracellular receptor domains, we found that although cytosolic domains from MERTK (CARMERTK) did not trigger antigen-specific cellular phagocytosis or killing effects, unlike those from MEGF10, FcRγ and CD3ζ did, these CARs all mediated similar SARS-CoV-2 clearance in vitro. Notably, we showed that CARMERTK macrophages reduced the virion load without upregulation of proinflammatory cytokine expression. These results suggest that CARMERTK drives an 'immunologically silent' scavenger effect in macrophages and pave the way for further investigation of CARs for the treatment of individuals with COVID-19, particularly those with severe cases at a high risk of hyperinflammation.


Subject(s)
COVID-19/drug therapy , COVID-19/immunology , Immunotherapy, Adoptive , Macrophages/immunology , SARS-CoV-2/immunology , Virion/immunology , Animals , COVID-19/genetics , Chlorocebus aethiops , Humans , Phagocytosis , SARS-CoV-2/genetics , THP-1 Cells , Vero Cells , Virion/genetics
8.
Med Princ Pract ; 30(5): 422-429, 2021.
Article in English | MEDLINE | ID: covidwho-1197290

ABSTRACT

Coronavirus disease 2019 (COVID-19), a pandemic infection with profound effects on human society, has challenged our ability to control viral infections. Although at the beginning of the COVID-19 outbreak, the epidemic seemed controllable in Southern Iran, the disease presented a critical pattern as of May 2020. After a few months of the emergence of COVID-19, its severity and mortality increased dramatically. It has been proposed that antibodies produced during previous exposure to local circulating human coronaviruses or possibly severe acute respiratory syndrome coronavirus 2 might contribute to the development of more severe and lethal presentations of COVID-19 possibly by triggering antibody-dependent enhancement. The binding of virions complexed with antibodies to Fcγ receptors on the target cells initiates receptor-mediated signaling events, leading to enhanced expression of inflammatory cytokines and suppression of intracellular antiviral responses at the transcriptome level, followed by endocytosis of the virus and subsequent activation of immune cells. The activated immune cells might accumulate in the lung and promote cytokine storm and lymphopenia. Furthermore, the formation of immune complexes can promote complement activation and subsequent tissue damage. Although there are currently no clinical data to support this hypothesis, a better understanding of these immunopathologic phenomena and their relation to the disease course and severity might give insights into the development of the most efficient prophylactic and therapeutic approaches. This review demonstrates the critical pattern of COVID-19 in Southern Iran and highlights the possible interplay of factors leading to this condition.


Subject(s)
Antibody-Dependent Enhancement/immunology , COVID-19/immunology , Pneumonia, Viral/immunology , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Cytokine Release Syndrome/immunology , Humans , Iran , Lymphopenia/immunology , Pandemics , Pneumonia, Viral/virology , Virion/immunology
9.
Emerg Microbes Infect ; 10(1): 774-781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1172617

ABSTRACT

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/virology , Immunity, Humoral , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cohort Studies , Coronavirus Nucleocapsid Proteins/immunology , Female , Follow-Up Studies , Germany , Humans , Immunoglobulin G/blood , Male , Middle Aged , Pandemics , Phosphoproteins/immunology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Virion/immunology , Young Adult
10.
Immunology ; 163(4): 416-430, 2021 08.
Article in English | MEDLINE | ID: covidwho-1142903

ABSTRACT

The sudden outbreak of SARS-CoV-2-infected disease (COVID-19), initiated from Wuhan, China, has rapidly grown into a global pandemic. Emerging evidence has implicated extracellular vesicles (EVs), a key intercellular communicator, in the pathogenesis and treatment of COVID-19. In the pathogenesis of COVID-19, cells that express ACE2 and CD9 can transfer these viral receptors to other cells via EVs, making recipient cells more susceptible for SARS-CoV-2 infection. Once infected, cells release EVs packaged with viral particles that further facilitate viral spreading and immune evasion, aggravating COVID-19 and its complications. In contrast, EVs derived from stem cells, especially mesenchymal stromal/stem cells, alleviate severe inflammation (cytokine storm) and repair damaged lung cells in COVID-19 by delivery of anti-inflammatory molecules. These therapeutic beneficial EVs can also be engineered into drug delivery platforms or vaccines to fight against COVID-19. Therefore, EVs from diverse sources exhibit distinct effects in regulating viral infection, immune response, and tissue damage/repair, functioning as a double-edged sword in COVID-19. Here, we summarize the recent progress in understanding the pathological roles of EVs in COVID-19. A comprehensive discussion of the therapeutic effects/potentials of EVs is also provided.


Subject(s)
COVID-19/virology , Extracellular Vesicles/virology , Lung/virology , Mesenchymal Stem Cells/metabolism , SARS-CoV-2/pathogenicity , Virion/metabolism , Animals , Antiviral Agents/administration & dosage , COVID-19/drug therapy , COVID-19/immunology , COVID-19/metabolism , COVID-19/therapy , COVID-19 Vaccines/administration & dosage , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/virology , Cytokines/metabolism , Drug Carriers , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Extracellular Vesicles/transplantation , Host-Pathogen Interactions , Humans , Inflammation Mediators/metabolism , Lung/immunology , Lung/metabolism , Mesenchymal Stem Cells/immunology , SARS-CoV-2/immunology , Virion/immunology
11.
Virus Res ; 295: 198305, 2021 04 02.
Article in English | MEDLINE | ID: covidwho-1065651

ABSTRACT

In this study, we showed that a codon optimized version of the spike (S) protein of SARS-CoV-2 can migrate to the cell membrane. However, efficient production of Moloney murine leukemia (MLV) infectious viral particles was only achieved with stable expression of a shorter S version in C-terminal (ΔS) in MLV Gag-pol expressing cells. As compared to transient transfections, this platform generated viruses with a 1000-fold higher titer. ΔS was 15-times more efficiently incorporated into VLPs as compared to S, and that was not due to steric interference between the cytoplasmic tail and the MLV capsid, as similar differences were also observed with extracellular vesicles. The amount of ΔS incorporated into VLPs released from producer cells was high and estimated at 1.25 µg/mL S2 equivalent (S is comprised of S1 and S2). The resulting VLPs could potentially be used alone or as a boost of other immunization strategies for COVID-19.


Subject(s)
COVID-19 Vaccines/immunology , Spike Glycoprotein, Coronavirus/biosynthesis , Virion/genetics , Cell Line , Humans , Moloney murine leukemia virus/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/immunology , Virion/immunology
12.
Biochem Biophys Res Commun ; 543: 45-49, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1053221

ABSTRACT

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.


Subject(s)
COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Coronavirus M Proteins/genetics , Coronavirus M Proteins/immunology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Immune Sera/immunology , Immunity, Humoral , Immunoblotting , Immunoglobulin G/blood , Phosphoproteins/genetics , Phosphoproteins/immunology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology
13.
Emerg Microbes Infect ; 9(1): 2653-2662, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-977352

ABSTRACT

In the face of COVID-19 pandemic caused by the newly emerged SARS-CoV-2, an inactivated, Vero cell-based, whole virion vaccine candidate has been developed and entered into phase III clinical trials within six months. Biochemical and immunogenic characterization of structural proteins and their post-translational modifications in virions, the end-products of the vaccine candidate, would be essential for the quality control and process development of vaccine products and for studying the immunogenicity and pathogenesis of SARS-CoV-2. By using a panel of rabbit antisera against virions and five structural proteins together with a convalescent serum, the spike (S) glycoprotein was shown to be N-linked glycosylated, PNGase F-sensitive, endoglycosidase H-resistant and cleaved by Furin-like proteases into S1 and S2 subunits. The full-length S and S1/S2 subunits could form homodimers/trimers. The membrane (M) protein was partially N-linked glycosylated; the accessory protein 3a existed in three different forms, indicative of cleavage and dimerization. Furthermore, analysis of the antigenicity of these proteins and their post-translationally modified forms demonstrated that S protein induced the strongest antibody response in both convalescent and immunized animal sera. Interestingly, immunization with the inactivated vaccine did not elicit antibody response against the S2 subunit, whereas strong antibody response against both S1 and S2 subunits was detected in the convalescent serum. Moreover, vaccination stimulated stronger antibody response against S multimers than did the natural infection. This study revealed that the native S glycoprotein stimulated neutralizing antibodies, while bacterially-expressed S fragments did not. The study on S modifications would facilitate design of S-based anti-SARS-CoV-2 vaccines.


Subject(s)
COVID-19 Vaccines , Protein Processing, Post-Translational , SARS-CoV-2/isolation & purification , Viral Structural Proteins , Virion , Animals , Antigens, Viral/analysis , Antigens, Viral/metabolism , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Cattle , Chlorocebus aethiops , Humans , Rabbits , SARS-CoV-2/immunology , Vaccines, Inactivated/chemistry , Vaccines, Inactivated/immunology , Vero Cells , Viral Structural Proteins/chemistry , Viral Structural Proteins/immunology , Viral Structural Proteins/isolation & purification , Virion/chemistry , Virion/immunology , Virion/isolation & purification
15.
Signal Transduct Target Ther ; 5(1): 212, 2020 09 22.
Article in English | MEDLINE | ID: covidwho-786590

ABSTRACT

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Severe Acute Respiratory Syndrome/prevention & control , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Betacoronavirus/drug effects , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cross Reactions , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/immunology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Protein Binding , Protein Structure, Secondary , Receptors, Virus/chemistry , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS Virus/drug effects , SARS Virus/immunology , SARS Virus/pathogenicity , SARS-CoV-2 , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/immunology , Virion/ultrastructure
16.
Anal Bioanal Chem ; 412(28): 7685-7699, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-737989

ABSTRACT

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , High-Throughput Screening Assays/instrumentation , Lab-On-A-Chip Devices , Microbial Sensitivity Tests/instrumentation , Animals , Bacterial Infections/drug therapy , Bacterial Infections/immunology , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/immunology , Cytokines/immunology , Drug Discovery/instrumentation , Drug Discovery/methods , Equipment Design , High-Throughput Screening Assays/methods , Humans , Microbial Sensitivity Tests/methods , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/immunology , Small Molecule Libraries/pharmacology , Virion/drug effects , Virion/immunology , Virus Diseases/drug therapy , Virus Diseases/immunology
17.
J Virol ; 94(21)2020 10 14.
Article in English | MEDLINE | ID: covidwho-709870

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Spike Glycoprotein, Coronavirus/physiology , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/metabolism , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , HEK293 Cells , HIV-1/genetics , HIV-1/metabolism , Humans , Leukemia Virus, Murine , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/metabolism , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Virion/genetics , Virion/immunology , Virion/metabolism , Virus Internalization
18.
Emerg Microbes Infect ; 9(1): 1712-1721, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-632216

ABSTRACT

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.


Subject(s)
Antigens, Viral/biosynthesis , Betacoronavirus/immunology , Proteomics/methods , Viral Vaccines/immunology , Virion/immunology , Animals , Antigens, Viral/immunology , Chlorocebus aethiops , SARS-CoV-2 , Tandem Mass Spectrometry , Vero Cells
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