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1.
Cancer Discov ; 12(4): 892-894, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1775023

ABSTRACT

SUMMARY: Fahrner and colleagues investigated the immune response of patients with cancer and cancer-free individuals to SARS-CoV-2 and found that a propensity toward an IL5-predominant Th2/Tc2 response was predictive of susceptibility to infection. The results of this study also suggest that a cellular response against the Spike 1 protein receptor binding domain (S1-RBD) region of the SARS-CoV-2 proteome contributes to protection and that mutations in this region may drive viral evolution and immune escape. See related article by Fahrner et al., p. 958 (8).


Subject(s)
COVID-19 , COVID-19/genetics , Humans , Membrane Glycoproteins/genetics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism
2.
PLoS Comput Biol ; 17(12): e1009664, 2021 12.
Article in English | MEDLINE | ID: covidwho-1571973

ABSTRACT

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise.


Subject(s)
Evolution, Molecular , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/biosynthesis , Antigens, Viral/chemistry , Antigens, Viral/genetics , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Computational Biology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Immune Evasion/genetics , Influenza, Human/immunology , Influenza, Human/virology , Models, Immunological , Molecular Dynamics Simulation , Mutation , Pandemics , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry
3.
Viruses ; 13(10)2021 09 28.
Article in English | MEDLINE | ID: covidwho-1481007

ABSTRACT

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.


Subject(s)
Antibodies, Viral/blood , Henipavirus Infections/prevention & control , Nipah Virus/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Respiratory Syncytial Virus Vaccines/administration & dosage , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/genetics , Viral Fusion Proteins/immunology
4.
Viruses ; 13(10)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470996

ABSTRACT

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1ß. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1ß release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.


Subject(s)
Immunity, Innate/immunology , Inflammasomes/immunology , Macrophages/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Viral Envelope Proteins/immunology , Viral Fusion Proteins/immunology , Cell Line, Tumor , Cytomegalovirus/immunology , Hepacivirus/immunology , Humans , Interleukin-1beta/biosynthesis , Interleukin-1beta/immunology , Pyroptosis/immunology , SARS Virus/immunology , SARS-CoV-2/immunology , THP-1 Cells
5.
Viruses ; 13(7)2021 06 30.
Article in English | MEDLINE | ID: covidwho-1287278

ABSTRACT

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.


Subject(s)
Host-Pathogen Interactions , Membrane Proteins/genetics , Viral Envelope Proteins , Virion/metabolism , Viruses/metabolism , HEK293 Cells , HIV-1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Membrane Proteins/immunology , Retroviridae/classification , Retroviridae/metabolism , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Virion/genetics , Virus Internalization , Viruses/chemistry , Viruses/classification , Viruses/genetics
6.
Viruses ; 13(6)2021 05 29.
Article in English | MEDLINE | ID: covidwho-1282636

ABSTRACT

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.


Subject(s)
Hepacivirus/chemistry , Hepatitis C/prevention & control , Viral Envelope Proteins/chemistry , Viral Hepatitis Vaccines/chemistry , Animals , Epitopes/immunology , HEK293 Cells , Hepacivirus/genetics , Hepacivirus/immunology , Hepatitis C/virology , Humans , Mice , Models, Molecular , Protein Conformation , Protein Multimerization , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism
7.
Microbiol Immunol ; 64(1): 33-51, 2020 Jan.
Article in English | MEDLINE | ID: covidwho-1262996

ABSTRACT

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


Subject(s)
Adjuvants, Immunologic/pharmacology , Coronavirus Infections/prevention & control , Gold/chemistry , Immunoglobulin G/immunology , Lung/immunology , Metal Nanoparticles/chemistry , Severe Acute Respiratory Syndrome/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Analysis of Variance , Animals , Antibodies, Viral/immunology , Chlorocebus aethiops , Coronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cytokines/metabolism , Disease Models, Animal , Female , Immunization , Lung/pathology , Mice , Mice, Inbred BALB C , Recombinant Proteins/immunology , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/genetics , Toll-Like Receptors , Vaccination , Vaccines, Synthetic , Vero Cells , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Viral Vaccines/pharmacology , Viral Vaccines/therapeutic use
8.
J Med Virol ; 93(1): 351-356, 2021 01.
Article in English | MEDLINE | ID: covidwho-1206800

ABSTRACT

Glycoproteins of enveloped viruses replicating in nonprimate mammalian cells carry α-1,3-galactose (α-Gal) glycans, and can bind to anti-Gal antibodies which are abundant in humans. The antibodies have protected humans and their ancestors for millions of years, because they inhibit replication of many kinds of microbes carrying αGal glycans and aid complements and macrophages to destroy them. Therefore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicating in nonprimate mammalian cells (eg, PK-15 cells) carry αGal glycans and could be employed as a live vaccine for corona virus 2019 (COVID-19). The live vaccine safety could be further enhanced through intramuscular inoculation to bypass the fragile lungs, like the live unattenuated adenovirus vaccine safely used in US recruits for decades. Moreover, the immune complexes of SARS-CoV-2 and anti-Gal antibodies could enhance the efficacy of COVID-19 vaccines, live or inactivated, carrying α-Gal glycans. Experiments are imperatively desired to examine these novel vaccine strategies which probably have the critical advantages for defeating the pandemic of COVID-19 and preventing other viral infectious diseases.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/physiology , Viral Envelope Proteins/immunology , Animals , Cell Line , Humans
9.
Mol Ther ; 29(3): 1174-1185, 2021 03 03.
Article in English | MEDLINE | ID: covidwho-985497

ABSTRACT

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.


Subject(s)
Immunity, Innate/drug effects , Immunogenicity, Vaccine , Protein Biosynthesis/drug effects , Vaccines, Synthetic/pharmacology , Viral Envelope Proteins/administration & dosage , Animals , Cell Line , Encephalitis Virus, Venezuelan Equine/drug effects , Encephalitis Virus, Venezuelan Equine/immunology , Encephalitis Virus, Venezuelan Equine/pathogenicity , Fibroblasts , Gene Expression Regulation , HeLa Cells , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunoglobulin G/biosynthesis , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Janus Kinases/antagonists & inhibitors , Janus Kinases/genetics , Janus Kinases/immunology , Mice , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/pathogenicity , NF-kappa B/genetics , NF-kappa B/immunology , Nitriles , Parainfluenza Virus 5/drug effects , Parainfluenza Virus 5/immunology , Parainfluenza Virus 5/pathogenicity , Pyrazoles/pharmacology , Pyrimidines , Rabbits , Rabies virus/drug effects , Rabies virus/immunology , Rabies virus/pathogenicity , Rats , STAT Transcription Factors/antagonists & inhibitors , STAT Transcription Factors/genetics , STAT Transcription Factors/immunology , Signal Transduction , Vaccines, Synthetic/biosynthesis , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology
10.
Nat Commun ; 12(1): 776, 2021 02 03.
Article in English | MEDLINE | ID: covidwho-1062751

ABSTRACT

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


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19 Vaccines/genetics , Cell Line , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , RNA, Messenger/genetics , RNA, Messenger/immunology , RNA, Viral/genetics , RNA, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology
11.
PLoS One ; 15(12): e0244176, 2020.
Article in English | MEDLINE | ID: covidwho-992710

ABSTRACT

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-COV-2) is a significant threat to global health security. Till date, no completely effective drug or vaccine is available to cure COVID-19. Therefore, an effective vaccine against SARS-COV-2 is crucially needed. This study was conducted to design an effective multiepitope based vaccine (MEV) against SARS-COV-2. Seven highly antigenic proteins of SARS-COV-2 were selected as targets and different epitopes (B-cell and T-cell) were predicted. Highly antigenic and overlapping epitopes were shortlisted. Selected epitopes indicated significant interactions with the HLA-binding alleles and 99.93% coverage of the world's population. Hence, 505 amino acids long MEV was designed by connecting 16 MHC class I and eleven MHC class II epitopes with suitable linkers and adjuvant. MEV construct was non-allergenic, antigenic, stable and flexible. Furthermore, molecular docking followed by molecular dynamics (MD) simulation analyses, demonstrated a stable and strong binding affinity of MEV with human pathogenic toll-like receptors (TLR), TLR3 and TLR8. Finally, MEV codons were optimized for its in silico cloning into Escherichia coli K-12 system, to ensure its increased expression. Designed MEV in present study could be a potential candidate for further vaccine production process against COVID-19. However, to ensure its safety and immunogenic profile, the proposed MEV needs to be experimentally validated.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence/genetics , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/therapeutic use , Computational Biology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , SARS-CoV-2/pathogenicity , Viral Envelope Proteins/immunology
12.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-913103

ABSTRACT

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Subject(s)
Antigens, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Immunogenicity, Vaccine/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19 , COVID-19 Vaccines , Cell Line , Coronavirus Infections/immunology , Cryoelectron Microscopy , Enzyme-Linked Immunosorbent Assay , Humans , Macaca fascicularis , Mice , Mice, Inbred BALB C , Neutralization Tests , Peptidyl-Dipeptidase A/metabolism , Protein Domains/genetics , Protein Domains/immunology , SARS-CoV-2 , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Vaccination , Viral Envelope Proteins/immunology
13.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-910292

ABSTRACT

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.


Subject(s)
Hepacivirus/physiology , Viral Envelope Proteins/metabolism , Antibody Affinity , Gene Knockdown Techniques , HEK293 Cells , Hepacivirus/immunology , Hepatitis C/virology , Hepatitis C Antibodies/immunology , Hepatitis C Antigens/immunology , Hepatitis C Antigens/metabolism , Humans , Mannose/chemistry , Polysaccharides/chemistry , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , Tetraspanin 28/genetics , Tetraspanin 28/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/immunology
14.
Virol J ; 17(1): 165, 2020 10 29.
Article in English | MEDLINE | ID: covidwho-895011

ABSTRACT

BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/virology , Epitopes, B-Lymphocyte/immunology , Pneumonia, Viral/virology , Viral Envelope Proteins/immunology , Viral Matrix Proteins/immunology , COVID-19 , COVID-19 Vaccines , Computational Biology , Coronavirus Envelope Proteins , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Humans , Immunogenicity, Vaccine/immunology , Models, Molecular , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/chemistry , Viral Vaccines/immunology
15.
Emerg Microbes Infect ; 9(1): 2361-2367, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-894519

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39 µg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Envelope Proteins/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/epidemiology , Female , Humans , Immunization , Male , Mice , Mice, Inbred BALB C , Pandemics , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Sequence Deletion , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/immunology , Virulence
16.
PLoS One ; 15(8): e0237300, 2020.
Article in English | MEDLINE | ID: covidwho-842269

ABSTRACT

The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H+ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct "core" structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.


Subject(s)
Betacoronavirus/chemistry , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Vaccines , Computer Simulation , Coronavirus Envelope Proteins , Coronavirus Infections/virology , Humans , Hydrogen , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Pneumonia, Viral/virology , Point Mutation , Protein Conformation , SARS-CoV-2 , Structural Homology, Protein , Vaccines, Attenuated , Vaccines, Inactivated , Viral Envelope Proteins/immunology , Viral Vaccines , Water/chemistry
17.
Life Sci ; 260: 118421, 2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-800912

ABSTRACT

In December 2019, a novel virus, namely COVID-19 caused by SARS-CoV-2, developed from Wuhan, (Hubei territory of China) used its viral spike glycoprotein receptor-binding domain (RBD) for the entrance into a host cell by binding with ACE-2 receptor and cause acute respiratory distress syndrome (ARDS). Data revealed that the newly emerged SARS-CoV-2 affected more than 24,854,140 people with 838,924 deaths worldwide. Until now, no licensed immunization or drugs are present for the medication of SARS-CoV-2. The present review aims to investigate the latest developments and discuss the candidate antibodies in different vaccine categories to develop a reliable and efficient vaccine against SARS-CoV-2 in a short time duration. Besides, the review focus on the present challenges and future directions, structure, and mechanism of SARS-CoV-2 for a better understanding. Based on data, we revealed that most of the vaccines are focus on targeting the spike protein (S) of COVID-19 to neutralized viral infection and develop long-lasting immunity. Up to phase-1 clinical trials, some vaccines showed the specific antigen-receptor T-cell response, elicit the humoral and immune response, displayed tight binding with human-leukocytes-antigen (HLA), and recognized specific antibodies to provoke long-lasting immunity against SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Betacoronavirus/immunology , Epitopes/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Humans , Pandemics/prevention & control , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , SARS-CoV-2
18.
Front Immunol ; 11: 2008, 2020.
Article in English | MEDLINE | ID: covidwho-781995

ABSTRACT

Coronavirus disease (COVID-19), caused by the virus SARS-CoV-2, is already responsible for more than 4.3 million confirmed cases and 295,000 deaths worldwide as of May 15, 2020. Ongoing efforts to control the pandemic include the development of peptide-based vaccines and diagnostic tests. In these approaches, HLA allelic diversity plays a crucial role. Despite its importance, current knowledge of HLA allele frequencies in South America is very limited. In this study, we have performed a literature review of datasets reporting HLA frequencies of South American populations, available in scientific literature and/or in the Allele Frequency Net Database. This allowed us to enrich the current scenario with more than 12.8 million data points. As a result, we are presenting updated HLA allelic frequencies based on country, including 91 alleles that were previously thought to have frequencies either under 5% or of an unknown value. Using alleles with an updated frequency of at least ≥5% in any South American country, we predicted epitopes in SARS-CoV-2 proteins using NetMHCpan (I and II) and MHC flurry. Then, the best predicted epitopes (class-I and -II) were selected based on their binding to South American alleles (Coverage Score). Class II predicted epitopes were also filtered based on their three-dimensional exposure. We obtained 14 class-I and four class-II candidate epitopes with experimental evidence (reported in the Immune Epitope Database and Analysis Resource), having good coverage scores for South America. Additionally, we are presenting 13 HLA-I and 30 HLA-II novel candidate epitopes without experimental evidence, including 16 class-II candidates in highly exposed conserved areas of the NTD and RBD regions of the Spike protein. These novel candidates have even better coverage scores for South America than those with experimental evidence. Finally, we show that recent similar studies presenting candidate epitopes also predicted some of our candidates but discarded them in the selection process, resulting in candidates with suboptimal coverage for South America. In conclusion, the candidate epitopes presented provide valuable information for the development of epitope-based strategies against SARS-CoV-2, such as peptide vaccines and diagnostic tests. Additionally, the updated HLA allelic frequencies provide a better representation of South America and may impact different immunogenetic studies.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Epitopes, T-Lymphocyte/immunology , Gene Frequency , HLA Antigens/genetics , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Viral Envelope Proteins/immunology , Alleles , Amino Acid Sequence , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Genetic Variation , Humans , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , SARS-CoV-2 , South America/epidemiology , Vaccines, Subunit/immunology , Viral Vaccines/immunology
19.
Emerg Microbes Infect ; 9(1): 2091-2093, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-759868

ABSTRACT

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ∼5-fold and ∼7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/immunology , Nucleocapsid/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19 , Coronavirus Infections/immunology , Female , Humans , Male , Middle Aged , Neutralization Tests , Pandemics , Pneumonia, Viral/immunology , SARS-CoV-2 , Severity of Illness Index , Viral Envelope Proteins/immunology
20.
JCI Insight ; 5(19)2020 10 02.
Article in English | MEDLINE | ID: covidwho-737501

ABSTRACT

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.


Subject(s)
Antibodies, Neutralizing/immunology , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Pneumonia, Viral/immunology , Pneumonia, Viral/therapy , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , Area Under Curve , COVID-19 , Enzyme-Linked Immunosorbent Assay , Humans , Immunization, Passive/methods , Neutralization Tests , Pandemics , Regression Analysis , Sampling Studies , Treatment Outcome , Viral Envelope Proteins/immunology
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