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1.
J Immunol Methods ; 502: 113216, 2022 03.
Article in English | MEDLINE | ID: covidwho-1611844

ABSTRACT

Coronavirus Disease 2019 (COVID-19) represents a new global threat demanding a multidisciplinary effort to fight its etiological agent-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this regard, immunoinformatics may aid to predict prominent immunogenic regions from critical SARS-CoV-2 structural proteins, such as the spike (S) glycoprotein, for their use in prophylactic or therapeutic interventions against this highly pathogenic betacoronavirus. Accordingly, in this study, an integrated immunoinformatics approach was applied to identify cytotoxic T cell (CTC), T helper cell (THC), and Linear B cell (BC) epitopes from the S glycoprotein in an attempt to design a high-quality multi-epitope vaccine. The best CTC, THC, and BC epitopes showed high viral antigenicity and lack of allergenic or toxic residues, as well as CTC and THC epitopes showed suitable interactions with HLA class I (HLA-I) and HLA class II (HLA-II) molecules, respectively. Remarkably, SARS-CoV-2 receptor-binding domain (RBD) and its receptor-binding motif (RBM) harbour several potential epitopes. The structure prediction, refinement, and validation data indicate that the multi-epitope vaccine has an appropriate conformation and stability. Four conformational epitopes and an efficient binding between Toll-like receptor 4 (TLR4) and the vaccine model were observed. Importantly, the population coverage analysis showed that the multi-epitope vaccine could be used globally. Notably, computer-based simulations suggest that the vaccine model has a robust potential to evoke and maximize both immune effector responses and immunological memory to SARS-CoV-2. Further research is needed to accomplish with the mandatory international guidelines for human vaccine formulations.


Subject(s)
Antigens, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Antigens, Viral/genetics , Antigens, Viral/metabolism , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/therapeutic use , Computational Biology , Computer Simulation , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Humans , Immunogenicity, Vaccine/genetics , Immunologic Memory , Protein Domains/genetics , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes, Cytotoxic , Toll-Like Receptor 4/metabolism , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/therapeutic use
2.
EBioMedicine ; 75: 103796, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587924

ABSTRACT

BACKGROUND: Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration. These two fundamental parameters cannot be disentangled in clinical immunoassays, and so produce a bias which depends on the assay format. METHODS: To determine the apparent affinity of anti-spike protein antibodies, a small number of antigen-coated magnetic microparticles were imaged by fluorescence microscopy after probing antigen-antibody equilibria directly in patient plasma. Direct and indirect anti-SARS-CoV-2 immunoassays were used to measure antibody levels in the blood of infected and immunised individuals. FINDINGS: We observed affinity maturation of antibodies in convalescent and vaccinated individuals, showing that higher affinities are achieved much faster by vaccination. We demonstrate that direct and indirect immunoassays for measuring anti-spike protein antibodies depend differently on antibody affinity which, in turn, affects accurate interpretation of the results. INTERPRETATION: Direct immunoassays show substantial antibody affinity dependence. This makes them useful for identifying past SARS-CoV-2 exposure. Indirect immunoassays provide more accurate quantifications of anti-viral antibody levels. FUNDING: The authors are all full-time employees of Abbott Laboratories. Abbott Laboratories provided all operating funds. No external funding sources were used in this study.


Subject(s)
Antibodies, Viral/immunology , Antibody Affinity , Antigens, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , Antigens, Viral/metabolism , COVID-19/blood , Humans , Immunoassay , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
3.
Biotechnol Lett ; 44(1): 45-57, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1536319

ABSTRACT

After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.


Subject(s)
COVID-19 Vaccines/biosynthesis , Plants, Genetically Modified/metabolism , SARS-CoV-2/immunology , Vaccines, Virus-Like Particle/biosynthesis , Antigens, Viral/genetics , Antigens, Viral/metabolism , COVID-19 Vaccines/economics , COVID-19 Vaccines/genetics , Gene Expression , Plants, Genetically Modified/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vaccines, Virus-Like Particle/economics , Vaccines, Virus-Like Particle/genetics , Viral Vaccines/biosynthesis , Viral Vaccines/genetics
4.
Sci Rep ; 11(1): 21460, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1500518

ABSTRACT

Population screening played a substantial role in safely reopening the economy and avoiding new outbreaks of COVID-19. PCR-based pooled screening makes it possible to test the population with limited resources by pooling multiple individual samples. Our study compared different population-wide screening methods as transmission-mitigating interventions, including pooled PCR, individual PCR, and antigen screening. Incorporating testing-isolation process and individual-level viral load trajectories into an epidemic model, we further studied the impacts of testing-isolation on test sensitivities. Results show that the testing-isolation process could maintain a stable test sensitivity during the outbreak by removing most infected individuals, especially during the epidemic decline. Moreover, we compared the efficiency, accuracy, and cost of different screening methods during the pandemic. Our results show that PCR-based pooled screening is cost-effective in reversing the pandemic at low prevalence. When the prevalence is high, PCR-based pooled screening may not stop the outbreak. In contrast, antigen screening with sufficient frequency could reverse the epidemic, despite the high cost and the large numbers of false positives in the screening process.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , Antigens, Viral/genetics , Antigens, Viral/metabolism , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , False Negative Reactions , False Positive Reactions , Humans , Pandemics , Polymerase Chain Reaction/economics , Reproducibility of Results , SARS-CoV-2/isolation & purification , Viral Load
5.
Virol J ; 18(1): 149, 2021 07 18.
Article in English | MEDLINE | ID: covidwho-1496197

ABSTRACT

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.


Subject(s)
COVID-19/pathology , Nasopharynx/ultrastructure , SARS-CoV-2/ultrastructure , Antigens, Viral/metabolism , COVID-19/diagnosis , COVID-19/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Humans , Image Enhancement , Microscopy , Microvilli/ultrastructure , Nasal Mucosa/ultrastructure , Nasal Mucosa/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Virion/ultrastructure
6.
Nature ; 583(7818): 834-838, 2020 07.
Article in English | MEDLINE | ID: covidwho-1387423

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/transmission , Coronavirus Infections/virology , Disease Models, Animal , Lung/pathology , Lung/virology , Mesocricetus/virology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Aerosols , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Antigens, Viral/metabolism , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , Bronchi/pathology , Bronchi/virology , COVID-19 , Coronavirus Infections/immunology , Duodenum/virology , Fomites/virology , Housing, Animal , Kidney/virology , Male , Mesocricetus/immunology , Nasal Mucosa/virology , Pandemics , Pneumonia, Viral/immunology , RNA, Viral/analysis , SARS-CoV-2 , Viral Load , Weight Loss
7.
J Proteome Res ; 19(11): 4398-4406, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-1387124

ABSTRACT

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.


Subject(s)
Aminopeptidases/metabolism , Antigens, Viral , Epitopes , Spike Glycoprotein, Coronavirus , Antigens, Viral/chemistry , Antigens, Viral/metabolism , Chromatography, Liquid , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , HLA Antigens/chemistry , HLA Antigens/metabolism , Humans , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Proteomics/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Tandem Mass Spectrometry
8.
Clin Immunol ; 231: 108828, 2021 10.
Article in English | MEDLINE | ID: covidwho-1363931

ABSTRACT

COVID-19 is characterized by a dysregulation of inflammatory cytokines ultimately resulting a cytokine storm that can result in significant morbidity and mortality. We developed an in-vitro assay using activated peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) or CD3 + CD28 to examine secretion of cytokines from antigen presenting cells (APCs) and T cells, respectively, in donor patients with a history of COVID-19 (convalescent) and uninfected negative controls. We hypothesized that a novel antioxidant called Tempol may decrease cytokines from activated peripheral blood cells from both COVID-19 patients and normal donors. Preincubation of immune cells with Tempol resulted in a significant (P < 0.05) decrease in multiple T cell and APC-derived cytokines from both cells of COVID-19 (n = 7) and uninfected donors (n = 7). These preliminary results suggest that Tempol has strong in-vitro anti-cytokine activity and supports additional studies examining the use of Tempol for the treatment of COVID-19.


Subject(s)
Antioxidants/pharmacology , COVID-19/immunology , Cyclic N-Oxides/pharmacology , Lymphocyte Activation/drug effects , SARS-CoV-2 , T-Lymphocytes/drug effects , Adult , Aged , Antigen-Presenting Cells/metabolism , Antigens, Viral/metabolism , Cytokines/antagonists & inhibitors , Cytokines/drug effects , Female , Humans , Male , Middle Aged , Spin Labels , T-Lymphocytes/physiology
9.
Viruses ; 13(6)2021 06 14.
Article in English | MEDLINE | ID: covidwho-1270128

ABSTRACT

Humoral immunity has emerged as a vital immune component against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nevertheless, a subset of recovered Coronavirus Disease-2019 (COVID-19) paucisymptomatic/asymptomatic individuals do not generate an antibody response, constituting a paradox. We assumed that immunodiagnostic assays may operate under a competitive format within the context of antigenemia, potentially explaining this phenomenon. We present a case where persistent antigenemia/viremia was documented for at least 73 days post-symptom onset using 'in-house' methodology, and as it progressively declined, seroconversion took place late, around day 55, supporting our hypothesis. Thus, prolonged SARS-CoV-2 antigenemia/viremia could mask humoral responses, rendering, in certain cases, the phenomenon of 'non-responders' a misnomer.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/blood , Antigens, Viral/immunology , COVID-19 Serological Testing/standards , COVID-19/diagnosis , SARS-CoV-2/immunology , Antibodies, Viral/metabolism , Antigens, Viral/metabolism , Binding Sites, Antibody , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/statistics & numerical data , Humans , Immunity, Humoral/immunology , Immunoglobulin G/blood , Male , Sensitivity and Specificity , Seroconversion , Young Adult
10.
Cell Rep ; 35(13): 109305, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1260679

ABSTRACT

The human leukocyte antigen (HLA)-bound viral antigens serve as an immunological signature that can be selectively recognized by T cells. As viruses evolve by acquiring mutations, it is essential to identify a range of presented viral antigens. Using HLA peptidomics, we are able to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides presented by highly prevalent HLA class I (HLA-I) molecules by using infected cells as well as overexpression of SARS-CoV-2 genes. We find 26 HLA-I peptides and 36 HLA class II (HLA-II) peptides. Among the identified peptides, some are shared between different cells and some are derived from out-of-frame open reading frames (ORFs). Seven of these peptides were previously shown to be immunogenic, and we identify two additional immunoreactive peptides by using HLA multimer staining. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on presented viral-specific antigens that span several of the viral genes.


Subject(s)
Antigens, Viral/immunology , COVID-19/immunology , COVID-19/virology , Peptides/immunology , SARS-CoV-2/immunology , Antigen Presentation , Antigens, Viral/metabolism , COVID-19 Vaccines , Cell Line , Epitopes, T-Lymphocyte/immunology , HEK293 Cells , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Peptidomimetics , SARS-CoV-2/genetics , T-Lymphocytes
11.
Science ; 373(6556): 818-823, 2021 08 13.
Article in English | MEDLINE | ID: covidwho-1238481

ABSTRACT

Neutralizing antibodies (nAbs) elicited against the receptor binding site (RBS) of the spike protein of wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are generally less effective against recent variants of concern. RBS residues Glu484, Lys417, and Asn501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on angiotensin-converting enzyme 2 binding, as well as the effects of two of these mutations (K417N and E484K) on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternative binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Antigenic Variation , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/metabolism , Binding Sites , Binding Sites, Antibody , COVID-19/virology , Epitopes , Humans , Immune Evasion , Mutation , Protein Binding , Protein Domains , Receptors, Coronavirus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
12.
Nat Commun ; 12(1): 2506, 2021 05 04.
Article in English | MEDLINE | ID: covidwho-1216457

ABSTRACT

It is unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can directly infect human kidney, thus leading to acute kidney injury (AKI). Here, we perform a retrospective analysis of clinical parameters from 85 patients with laboratory-confirmed coronavirus disease 2019 (COVID-19); moreover, kidney histopathology from six additional COVID-19 patients with post-mortem examinations was performed. We find that 27% (23/85) of patients exhibited AKI. The elderly patients and cases with comorbidities (hypertension and heart failure) are more prone to develop AKI. Haematoxylin & eosin staining shows that the kidneys from COVID-19 autopsies have moderate to severe tubular damage. In situ hybridization assays illustrate that viral RNA accumulates in tubules. Immunohistochemistry shows nucleocapsid and spike protein deposits in the tubules, and immunofluorescence double staining shows that both antigens are restricted to the angiotensin converting enzyme-II-positive tubules. SARS-CoV-2 infection triggers the expression of hypoxic damage-associated molecules, including DP2 and prostaglandin D synthase in infected tubules. Moreover, it enhances CD68+ macrophages infiltration into the tubulointerstitium, and complement C5b-9 deposition on tubules is also observed. These results suggest that SARS-CoV-2 directly infects human kidney to mediate tubular pathogenesis and AKI.


Subject(s)
Acute Kidney Injury/etiology , COVID-19/complications , Kidney Tubules/virology , SARS-CoV-2/pathogenicity , Acute Kidney Injury/epidemiology , Acute Kidney Injury/pathology , Acute Kidney Injury/virology , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Antigens, Viral/genetics , Antigens, Viral/metabolism , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Female , Humans , Immunity, Innate , Kidney Function Tests , Kidney Tubules/metabolism , Kidney Tubules/pathology , Male , Middle Aged , Pandemics , Retrospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Proteins/genetics , Viral Proteins/metabolism , Young Adult
13.
J Phys Chem Lett ; 12(8): 2166-2171, 2021 Mar 04.
Article in English | MEDLINE | ID: covidwho-1101616

ABSTRACT

The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been determined to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.


Subject(s)
Antigens, Viral/analysis , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , COVID-19/diagnosis , Gold/chemistry , Metal Nanoparticles/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/analysis , Angiotensin-Converting Enzyme 2/metabolism , Antigens, Viral/metabolism , Aptamers, Nucleotide/metabolism , COVID-19 Testing/methods , Energy Transfer , Humans , Limit of Detection , Protein Binding , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism
14.
J Heart Lung Transplant ; 39(4): 379-388, 2020 04.
Article in English | MEDLINE | ID: covidwho-783270

ABSTRACT

BACKGROUND: Respiratory viral infections can increase the risk of chronic lung allograft dysfunction after lung transplantation, but the mechanisms are unknown. In this study, we determined whether symptomatic respiratory viral infections after lung transplantation induce circulating exosomes that contain lung-associated self-antigens and assessed whether these exosomes activate immune responses to self-antigens. METHODS: Serum samples were collected from lung transplant recipients with symptomatic lower- and upper-tract respiratory viral infections and from non-symptomatic stable recipients. Exosomes were isolated via ultracentrifugation; purity was determined using sucrose cushion; and presence of lung self-antigens, 20S proteasome, and viral antigens for rhinovirus, coronavirus, and respiratory syncytial virus were determined using immunoblot. Mice were immunized with circulating exosomes from each group and resulting differential immune responses and lung histology were analyzed. RESULTS: Exosomes containing self-antigens, 20S proteasome, and viral antigens were detected at significantly higher levels (p < 0.05) in serum of recipients with symptomatic respiratory viral infections (n = 35) as compared with stable controls (n = 32). Mice immunized with exosomes from recipients with respiratory viral infections developed immune responses to self-antigens, fibrosis, small airway occlusion, and significant cellular infiltration; mice immunized with exosomes from controls did not (p < 0.05). CONCLUSIONS: Circulating exosomes isolated from lung transplant recipients diagnosed with respiratory viral infections contained lung self-antigens, viral antigens, and 20S proteasome and elicited immune responses to lung self-antigens that resulted in development of chronic lung allograft dysfunction in immunized mice.


Subject(s)
Exosomes/metabolism , Graft Rejection/etiology , Graft Rejection/metabolism , Lung Transplantation/adverse effects , Respiratory Tract Infections/metabolism , Virus Diseases/metabolism , Aged , Animals , Antigens, Viral/metabolism , Autoantigens/metabolism , Case-Control Studies , Female , HLA Antigens/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Proteasome Endopeptidase Complex/metabolism , Respiratory Tract Infections/complications , Respiratory Tract Infections/virology , Virus Diseases/complications
15.
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
16.
Int J Mol Sci ; 21(23)2020 Dec 01.
Article in English | MEDLINE | ID: covidwho-954912

ABSTRACT

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.


Subject(s)
Antibodies, Monoclonal/metabolism , Antigens, Viral/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Antigens, Viral/immunology , COVID-19/pathology , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Hybridomas/cytology , Hybridomas/metabolism , Immunohistochemistry , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Protein Domains/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
17.
Avian Dis ; 64(2): 183-196, 2020 06.
Article in English | MEDLINE | ID: covidwho-892407

ABSTRACT

Nine infectious bronchitis virus (IBV) strains belonging to the GI-7 lineage were isolated between 2009 and 2017 in China. Phylogenetic analysis and comparisons of full-length sequences of the S1 gene suggested that the GI-7 lineage should be further classified as Taiwan (TW)-I and TW-II sublineages, which correspond to the previous TW-I and TW-II genotypes. The nine IBV strains were clustered in the TW-II sublineage. Further investigation revealed that viruses in the TW-I and TW-II were not only genetically but also antigenically different. Moreover, the TW-II sublineage contained various clades and recombinants. A recombinant was found to originate from recombination events between field strains (TW-II ck/CH/LJL/090608- and GI-19 ck/ CH/LDL/091022-like viruses) in which the recombination in the S1 subunit coding sequences had led to changes in antigenicity of the viruses. A more in-depth investigation demonstrated that TW-II viruses appear to have undergone a significant evolution following introduction in mainland China, which resulted in the viruses diverging into different clades. The viruses between the different clades in TW-II sublineage exhibited a significant change in genetic and antigenic characteristics. In addition, the five TW-II viruses selected on the basis of the results of S1 nucleotide sequence phylogenetic trees showed different pathogenicity to specific-pathogen-free chickens, although they could induce nephritis in the infected chickens and thus were identified as nephropathogenic strains.


Características genéticas, antigénicas y patógenas del virus de la bronquitis infecciosa GI-7/TW-II en China. Nueve cepas del virus de la bronquitis infecciosa (IBV) que pertenecen al linaje GI-7 se aislaron entre 2009 y 2017 en China. El análisis filogenético y las comparaciones de las secuencias completas del gene S1 sugirieron que el linaje GI-7 debería ser clasificado además como sublinajes TW-I y TW-II, que corresponden a los anteriores genotipos TW-T y TW-II. Las nueve cepas del virus de la bronquitis infecciosa se agruparon en el sublinaje TW-II. La investigación adicional reveló que los virus en TW-I y TW-II no solo eran tanto genéticamente como antigénicamente diferentes. Además, el sublinaje TW-II contenía varios clados y recombinantes. Se descubrió que un recombinante se originaba a partir de eventos de recombinación entre cepas de campo (virus similares a las cepas TW-II ck/CH/LJL/090608 y GI-19 ck/CH/LDL/091022) en los que la recombinación en las secuencias de codificación de la subunidad de S1 condujo a cambios en la antigenicidad de los virus. Una investigación más profunda demostró que los virus TW-II parecen haber experimentado una evolución significativa después de su introducción en China continental, lo que resultó en la divergencia de los virus en diferentes clados. Los virus entre los diferentes clados en el sublinaje TW-II exhibieron un cambio significativo en las características genéticas y antigénicas. Además, los cinco virus TW-II seleccionados con base en los resultados de los árboles filogenéticos de las secuencias de nucleótidos de S1 mostraron patogenicidad diferente en los pollos libres de patógenos específicos, aunque pudieron inducir nefritis en los pollos infectados y, por lo tanto, se identificaron como cepas nefropatógenas.


Subject(s)
Chickens , Coronavirus Infections/veterinary , Infectious bronchitis virus , Poultry Diseases/virology , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Antigens, Viral/genetics , Antigens, Viral/metabolism , China , Coronavirus Infections/virology , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Infectious bronchitis virus/pathogenicity , Phylogeny , Sequence Alignment , Specific Pathogen-Free Organisms , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
18.
mBio ; 11(5)2020 10 20.
Article in English | MEDLINE | ID: covidwho-883315

ABSTRACT

An accurate diagnostic test for early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the key weapon to control the coronavirus disease 2019 (COVID-19) pandemic. We previously reported that the SARS-CoV-2 genome contains a unique orf8 accessory gene absent from other human-pathogenic coronaviruses. Here, we characterized the SARS-CoV-2 orf8 as a novel immunogenic secreted protein and utilized it for the accurate diagnosis of COVID-19. Extracellular orf8 protein was detected in cell culture supernatant and in sera of COVID-19 patients. In addition, orf8 was found highly immunogenic in COVID-19 patients, who showed early seropositivity for anti-orf8 IgM, IgG, and IgA. We hypothesize that orf8 secretion during SARS-CoV-2 infection facilitates early mounting of B cell response. The serological test detecting anti-orf8 IgG antibody can be used for the early and accurate diagnosis of COVID-19.IMPORTANCE Current commercially available serological tests for COVID-19 patients are detecting antibodies against SARS-CoV-2 nucleoprotein and spike glycoprotein. The antinucleoprotein and antispike antibodies can be accurately detected in patients during the mid or late stage of infection, and therefore, these assays have not been widely used for early diagnosis of COVID-19. In this study, we characterized the secretory property of a SARS-CoV-2 orf8 protein and proposed that orf8 secretion during infection facilitates early mounting of the B cell response. We demonstrated the presence of anti-orf8 antibodies in both symptomatic and asymptomatic patients during the early stage of infection, while the anti-N antibody is not detected. Our serological test detecting anti-orf8 antibodies may facilitate the development of early and accurate diagnosis for COVID-19.


Subject(s)
Antigens, Viral/immunology , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Viral Proteins/immunology , Antibodies, Viral/blood , Antigens, Viral/blood , Antigens, Viral/metabolism , COVID-19 , Cell Line , Coronavirus Infections/blood , Early Diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/blood , Pandemics , Pneumonia, Viral/blood , SARS-CoV-2 , Viral Proteins/blood , Viral Proteins/metabolism
19.
Indian J Med Microbiol ; 38(2): 210-212, 2020.
Article in English | MEDLINE | ID: covidwho-745217

ABSTRACT

Recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent containment procedures have impacted the world as never seen before. Therefore, there is considerable curiosity about the genome evolution related to the origin, transmission and vaccine impact of this virus. We have analysed genome sequences of SARS-CoV-2 isolated from Indian patients to gain an in-depth understanding of genomic evolution and transmission in India. Phylogenetic analysis and mutation profiling revealed major lineages being evolved by characteristic mutations. As the mutation frequency in spike protein is comparatively lesser, the candidate vaccines expected to have wide coverage worldwide including India.


Subject(s)
Antigens, Viral/genetics , Betacoronavirus/genetics , Coronavirus Infections/prevention & control , Genome, Viral , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/genetics , Viral Vaccines/genetics , Animals , Antigens, Viral/immunology , Antigens, Viral/metabolism , Betacoronavirus/classification , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Chiroptera/virology , Coronavirus Infections/epidemiology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/transmission , Disease Reservoirs/virology , Eutheria/virology , Evolution, Molecular , Humans , India/epidemiology , Mutation , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/transmission , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Vaccines/biosynthesis , Viral Vaccines/immunology
20.
Vaccine ; 38(42): 6487-6499, 2020 09 29.
Article in English | MEDLINE | ID: covidwho-720733

ABSTRACT

The many carbohydrate chains on Covid-19 coronavirus SARS-CoV-2 and its S-protein form a glycan-shield that masks antigenic peptides and decreases uptake of inactivated virus or S-protein vaccines by APC. Studies on inactivated influenza virus and recombinant gp120 of HIV vaccines indicate that glycoengineering of glycan-shields to present α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) enables harnessing of the natural anti-Gal antibody for amplifying vaccine efficacy, as evaluated in mice producing anti-Gal. The α-gal epitope is the ligand for the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. Upon administration of vaccines presenting α-gal epitopes, anti-Gal binds to these epitopes at the vaccination site and forms immune complexes with the vaccines. These immune complexes are targeted for extensive uptake by APC as a result of binding of the Fc portion of immunocomplexed anti-Gal to Fc receptors on APC. This anti-Gal mediated effective uptake of vaccines by APC results in 10-200-fold higher anti-viral immune response and in 8-fold higher survival rate following challenge with a lethal dose of live influenza virus, than same vaccines lacking α-gal epitopes. It is suggested that glycoengineering of carbohydrate chains on the glycan-shield of inactivated SARS-CoV-2 or on S-protein vaccines, for presenting α-gal epitopes, will have similar amplifying effects on vaccine efficacy. α-Gal epitope synthesis on coronavirus vaccines can be achieved with recombinant α1,3galactosyltransferase, replication of the virus in cells with high α1,3galactosyltransferase activity as a result of stable transfection of cells with several copies of the α1,3galactosyltransferase gene (GGTA1), or by transduction of host cells with replication defective adenovirus containing this gene. In addition, recombinant S-protein presenting multiple α-gal epitopes on the glycan-shield may be produced in glycoengineered yeast or bacteria expression systems containing the corresponding glycosyltransferases. Prospective Covid-19 vaccines presenting α-gal epitopes may provide better protection than vaccines lacking this epitope because of increased uptake by APC.


Subject(s)
Antigens, Viral/genetics , Betacoronavirus/drug effects , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/genetics , Trisaccharides/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/biosynthesis , Antigens, Viral/immunology , Antigens, Viral/metabolism , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/virology , Genetic Engineering , HIV Core Protein p24/chemistry , HIV Core Protein p24/genetics , HIV Core Protein p24/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , Humans , Immunogenicity, Vaccine , Macrophages/drug effects , Macrophages/immunology , Macrophages/virology , Mice , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Trisaccharides/chemistry , Viral Vaccines/administration & dosage , Viral Vaccines/biosynthesis , Viral Vaccines/genetics
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