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1.
Methods Mol Biol ; 2410: 265-272, 2022.
Article in English | MEDLINE | ID: covidwho-1575755

ABSTRACT

COVID-19 caused by SARS-CoV-2, an RNA coronavirus has impacted the health and economy of all the countries. The virus has wide host adaptability and causes severe diseases in humans and animals. The major structural proteins of SARS-CoV-2 include spike (S), envelop (E), membrane (M), and nucleocapsid (N). The current vaccines are based on the S protein. The emergence of variants of SARS-CoV-2 has renewed interest in the use of additional structural proteins for the development of diagnostics and vaccines. Knowledge of B cell epitopes and MHC-I binding regions of the structural proteins of SARS-CoV-2 is essential in the development of effective diagnostics and therapies. This chapter provides information on the epitopes of the structural proteins of SARS-CoV-2.


Subject(s)
Coronavirus Envelope Proteins/immunology , Epitopes, B-Lymphocyte , Epitopes, T-Lymphocyte , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology , Animals , COVID-19 , COVID-19 Vaccines , Humans , SARS-CoV-2
2.
Sci Rep ; 11(1): 20383, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469988

ABSTRACT

SARS-CoV-2 continues to infect an ever-expanding number of people, resulting in an increase in the number of deaths globally. With the emergence of new variants, there is a corresponding decrease in the currently available vaccine efficacy, highlighting the need for greater insights into the viral epitope profile for both vaccine design and assessment. In this study, three immunodominant linear B cell epitopes in the SARS-CoV-2 spike receptor-binding domain (RBD) were identified by immunoinformatics prediction, and confirmed by ELISA with sera from Macaca fascicularis vaccinated with a SARS-CoV-2 RBD subunit vaccine. Further immunoinformatics analyses of these three epitopes gave rise to a method of linear B cell epitope prediction and selection. B cell epitopes in the spike (S), membrane (M), and envelope (E) proteins were subsequently predicted and confirmed using convalescent sera from COVID-19 infected patients. Immunodominant epitopes were identified in three regions of the S2 domain, one region at the S1/S2 cleavage site and one region at the C-terminus of the M protein. Epitope mapping revealed that most of the amino acid changes found in variants of concern are located within B cell epitopes in the NTD, RBD, and S1/S2 cleavage site. This work provides insights into B cell epitopes of SARS-CoV-2 as well as immunoinformatics methods for B cell epitope prediction, which will improve and enhance SARS-CoV-2 vaccine development against emergent variants.


Subject(s)
COVID-19/immunology , Epitopes, B-Lymphocyte/immunology , Immunodominant Epitopes/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology , Animals , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Computational Biology , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/immunology , Epitopes, B-Lymphocyte/chemistry , Humans , Immunoassay , Immunodominant Epitopes/chemistry , Macaca , Models, Molecular , Spike Glycoprotein, Coronavirus/chemistry , Viral Matrix Proteins/chemistry
3.
PLoS Pathog ; 17(9): e1009842, 2021 09.
Article in English | MEDLINE | ID: covidwho-1416911

ABSTRACT

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0-79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Acute Disease , Adult , Aged , Cohort Studies , Coronavirus Envelope Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunospot Assay , Epitopes, T-Lymphocyte/immunology , Female , Humans , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Survivors , T-Cell Antigen Receptor Specificity , Viral Matrix Proteins/immunology
4.
Front Immunol ; 12: 679841, 2021.
Article in English | MEDLINE | ID: covidwho-1369665

ABSTRACT

Understanding the course of the antibody response directed to individual epitopes of SARS-CoV-2 proteins is crucial for serological assays and establishment of vaccines. Twenty-one synthetic peptides were synthesized that have ten amino acids overlap and cover the complete membrane (M) protein. Plasma samples from 32 patients having acute disease and 30 patients from the convalescent phase were studied. Only peptide M01 (aa 1-20) and to a lesser extent peptide M21 (aa 201-222) showed specific reactivity as compared to historical control plasma samples. Peptide M01 was recognized by IgM- (71.9%) and IgG-specific antibodies (43.8%) during the acute phase as early as day 8 PIO. In a longitudinal analysis, a higher reactivity was observed for the IgM response directed to peptide M01 following day 20 PIO as compared to earlier time points of the acute phase. In the convalescent phase, antibody reactivity to the two M-specific peptides was significantly lower (<30% seropositivity). A fusion protein encoding major parts of RBD also showed higher rates of recognition during acute (50.0%) and lower rates in the convalescent phase (23.3%). Taken together, our results suggest that during the acute phase of COVID-19 antibodies are raised to two linear epitopes of the SARS-CoV-2 M protein, located at the very N- and C-termini, showing almost similar levels of reactivity as immunodominant linear epitopes derived from the spike and nucleocapsid protein. Anti-M is also present in the convalescent phase of COVID-19 patients, however at lower levels, with the N-terminus of the M protein as a preferred target.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Viral Matrix Proteins/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/biosynthesis , Convalescence , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunodominant Epitopes/immunology , Immunoglobulin M/immunology , Male , Middle Aged , Patient Acuity , Peptide Fragments/immunology , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/genetics
5.
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
6.
J Immunol ; 207(4): 1099-1111, 2021 08 15.
Article in English | MEDLINE | ID: covidwho-1328168

ABSTRACT

Understanding the function of SARS-CoV-2 Ag-specific T cells is crucial for the monitoring of antiviral immunity and vaccine design. Currently, both impaired and robust T cell immunity is described in COVID-19 patients. In this study, we explored and compared the effector functions of SARS-CoV-2-reactive T cells expressing coinhibitory receptors and examine the immunogenicity of SARS-CoV-2 S, M, and N peptide pools in regard to specific effector T cell responses, Th1/Th2/Th17, in COVID-19 patients. Analyzing a cohort of 108 COVID-19 patients with mild, moderate, and severe disease, we observed that coinhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, VISTA, CD39, CD160, 2B4, TIGIT, Gal-9, and NKG2A) were upregulated on both CD4+ and CD8+ T cells. Importantly, the expression of coinhibitory receptors on T cells recognizing SARS-CoV-2 peptide pools (M/N/S) was associated with increased frequencies of cytokine-producing T cells. Thus, our data refute the concept of pathological T cell exhaustion in COVID-19 patients. Despite interindividual variations in the T cell response to viral peptide pools, a Th2 phenotype was associated with asymptomatic and milder disease, whereas a robust Th17 was associated with severe disease, which may potentiate the hyperinflammatory response in patients admitted to the Intensive Care Unit. Our data demonstrate that T cells may either play a protective or detrimental role in COVID-19 patients. This finding could have important implications for immune correlates of protection, diagnostic, and prophylaxis with respect to COVID-19 management.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Th17 Cells/immunology , Th2 Cells/immunology , Adult , Aged , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Lymphocyte Activation/immunology , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Viral Matrix Proteins/immunology
7.
PLoS Pathog ; 17(7): e1009761, 2021 07.
Article in English | MEDLINE | ID: covidwho-1315898

ABSTRACT

T-cell immunity is likely to play a role in protection against SARS-CoV-2 by helping generate neutralizing antibodies. We longitudinally studied CD4 T-cell responses to the M, N, and S structural proteins of SARS-CoV-2 in 26 convalescent individuals. Within the first two months following symptom onset, a majority of individuals (81%) mounted at least one CD4 T-cell response, and 48% of individuals mounted detectable SARS-CoV-2-specific circulating T follicular helper cells (cTfh, defined as CXCR5+PD1+ CD4 T cells). SARS-CoV-2-specific cTfh responses across all three protein specificities correlated with antibody neutralization with the strongest correlation observed for S protein-specific responses. When examined over time, cTfh responses, particularly to the M protein, increased in convalescence, and robust cTfh responses with magnitudes greater than 5% were detected at the second convalescent visit, a median of 38 days post-symptom onset. CD4 T-cell responses declined but persisted at low magnitudes three months and six months after symptom onset. These data deepen our understanding of antigen-specific cTfh responses in SARS-CoV-2 infection, suggesting that in addition to S protein, M and N protein-specific cTfh may also assist in the development of neutralizing antibodies and that cTfh response formation may be delayed in SARS-CoV-2 infection.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , T Follicular Helper Cells/immunology , T Follicular Helper Cells/virology , Adult , Aged , Antibody Specificity , Case-Control Studies , Coronavirus Nucleocapsid Proteins/immunology , Female , Host Microbial Interactions/immunology , Humans , Longitudinal Studies , Male , Middle Aged , Pandemics , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Viral Matrix Proteins/immunology , Young Adult
8.
Front Immunol ; 12: 662989, 2021.
Article in English | MEDLINE | ID: covidwho-1256380

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of current COVID-19 pandemic, and insufficient production of type I interferon (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS-CoV-2 has been reported to suppress host IFN-I production, but the underlying mechanism is not completely understood. In this study, SARS-CoV-2 M protein was confirmed to suppress the expression of IFNß and interferon-stimulated genes induced by RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further impaired the formation of TRAF3-TANK-TBK1-IKKε complex that leads to inhibition of IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative regulation of IFN-I production, which would provide deeper insight into the innate immunosuppression and pathogenicity of SARS-CoV-2.


Subject(s)
Interferon Type I/biosynthesis , SARS-CoV-2/immunology , Ubiquitin/metabolism , Viral Matrix Proteins/immunology , DEAD Box Protein 58/metabolism , HEK293 Cells , Humans , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Proteolysis , Receptors, Immunologic/metabolism , Signal Transduction , TNF Receptor-Associated Factor 3/metabolism
9.
Int J Mol Sci ; 22(9)2021 May 07.
Article in English | MEDLINE | ID: covidwho-1224027

ABSTRACT

Diagnostic evaluation of specific antibodies against the SARS-CoV-2 virus is mainly based on spike (S) and nucleocapsid (N) proteins. Despite the critical functions in virus infection and contribution to the pattern of immunodominance in COVID-19, exploitation of the most abundant membrane (M) protein in the SARS-CoV-2 serology tests is minimal. This study investigated the recombinant M protein's immunoreactivity with the sera from COVID-19 convalescents. In silico designed protein was created from the outer N-terminal part (19 aa) and internal C-terminal tail (101-222 aa) of the M protein (YP_009724393.1) and was recombinantly produced and purified. The designed M protein (16,498.74 Da, pI 8.79) revealed both IgM and IgG reactivity with serum samples from COVID-19 convalescents in Western blot. In ELISA, more than 93% (28/30) of COVID-19 sera were positive for IgM detection, and more than 96% (29/30) were positive for specific IgG detection to M protein. Based on the capacity to provoke an immune response and its strong antigenic properties, as shown here, and the fact that it is also involved in the virion entry into host cells, the M protein of the SARS-CoV-2 virus as a good antigen has the potential in diagnostic purposes and vaccine design.


Subject(s)
COVID-19/blood , Immunoglobulin G/immunology , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Viral Matrix Proteins/immunology , COVID-19/immunology , Humans , Recombinant Proteins/immunology , Sensitivity and Specificity , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/isolation & purification
10.
Vaccine ; 39(7): 1111-1121, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1065643

ABSTRACT

At the beginning of the year 2020, the world was struck with a global pandemic virus referred to as SARS-CoV-2 (COVID-19) which has left hundreds of thousands of people dead. To control this virus, vaccine design becomes imperative. In this study, potential epitopes-based vaccine candidates were explored. Six hundred (600) genomes of SARS-CoV-2 were retrieved from the viPR database to generate CD8+ T-cell, CD4+ T-cell and linear B-cell epitopes which were screened for antigenicity, immunogenicity and non-allergenicity. The results of this study provide 19 promising candidate CD8+ T-cell epitopes that strongly overlap with 8 promising B-cells epitopes. Another 19 CD4+ T-cell epitopes were also identified that can induce IFN-γ and IL-4 cytokines. The most conserved MHC-I and MHC-II for both CD8+ and CD4+ T-cell epitopes are HLA-A*02:06 and HLA-DRB1*01:01 respectively. These epitopes also bound to Toll-like receptor 3 (TLR3). The population coverage of the conserved Major Histocompatibility Complex Human Leukocyte Antigen (HLA) for both CD8+ T-cell and CD4+ T-cell ranged from 65.6% to 100%. The detailed analysis of the potential epitope-based vaccine and their mapping to the complete COVID-19 genome reveals that they are predominantly found in the location of the surface (S) and membrane (M) glycoproteins suggesting the potential involvement of these structural proteins in the immunogenic response and antigenicity of the virus. Since the majority of the potential epitopes are located on M protein, the design of multi-epitope vaccine with the structural protein is highly promising though the whole M protein could also serve as a viable epitope for the development of an attenuated vaccine. Our findings provide a baseline for the experimental design of a suitable vaccine against SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Humans , Interferon-gamma , Interleukin-4 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology
11.
Cell Rep ; 34(4): 108666, 2021 01 26.
Article in English | MEDLINE | ID: covidwho-1064915

ABSTRACT

Although vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulate the 3D structures and predict the B cell epitopes on the spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches and validate epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induce antibody production, six of these are immunodominant epitopes in individuals, and 23 are conserved within SARS-CoV-2, SARS-CoV, and bat coronavirus RaTG13. We find that the immunodominant epitopes of individuals with domestic (China) SARS-CoV-2 are different from those of individuals with imported (Europe) SARS-CoV-2, which may be caused by mutations on the S (G614D) and N proteins. Importantly, we find several epitopes on the S protein that elicit neutralizing antibodies against D614 and G614 SARS-CoV-2, which can contribute to vaccine design against coronaviruses.


Subject(s)
Coronavirus Nucleocapsid Proteins/immunology , Epitopes, B-Lymphocyte/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology , Viroporin Proteins/immunology , Adolescent , Adult , Aged , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/therapy , COVID-19 Vaccines/immunology , Child , Epitopes, B-Lymphocyte/metabolism , Female , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Young Adult
12.
Int J Mol Sci ; 22(3)2021 Jan 28.
Article in English | MEDLINE | ID: covidwho-1055069

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a novel epidemic strain of Betacoronavirus that is responsible for the current viral pandemic, coronavirus disease 2019 (COVID-19), a global health crisis. Other epidemic Betacoronaviruses include the 2003 SARS-CoV-1 and the 2009 Middle East Respiratory Syndrome Coronavirus (MERS-CoV), the genomes of which, particularly that of SARS-CoV-1, are similar to that of the 2019 SARS-CoV-2. In this extensive review, we document the most recent information on Coronavirus proteins, with emphasis on the membrane proteins in the Coronaviridae family. We include information on their structures, functions, and participation in pathogenesis. While the shared proteins among the different coronaviruses may vary in structure and function, they all seem to be multifunctional, a common theme interconnecting these viruses. Many transmembrane proteins encoded within the SARS-CoV-2 genome play important roles in the infection cycle while others have functions yet to be understood. We compare the various structural and nonstructural proteins within the Coronaviridae family to elucidate potential overlaps and parallels in function, focusing primarily on the transmembrane proteins and their influences on host membrane arrangements, secretory pathways, cellular growth inhibition, cell death and immune responses during the viral replication cycle. We also offer bioinformatic analyses of potential viroporin activities of the membrane proteins and their sequence similarities to the Envelope (E) protein. In the last major part of the review, we discuss complement, stimulation of inflammation, and immune evasion/suppression that leads to CoV-derived severe disease and mortality. The overall pathogenesis and disease progression of CoVs is put into perspective by indicating several stages in the resulting infection process in which both host and antiviral therapies could be targeted to block the viral cycle. Lastly, we discuss the development of adaptive immunity against various structural proteins, indicating specific vulnerable regions in the proteins. We discuss current CoV vaccine development approaches with purified proteins, attenuated viruses and DNA vaccines.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/metabolism , Viral Matrix Proteins/metabolism , Animals , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19/immunology , COVID-19/metabolism , COVID-19/pathology , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Genome, Viral , Host-Pathogen Interactions , Humans , Immune Evasion , Protein Interaction Maps , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Virus Internalization , Virus Replication
13.
Nature ; 591(7850): 482-487, 2021 03.
Article in English | MEDLINE | ID: covidwho-1049967

ABSTRACT

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.


Subject(s)
Antibodies, Viral/analysis , Biosensing Techniques/methods , Hepatitis B virus/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/analysis , Troponin I/analysis , Antibodies, Viral/immunology , Biosensing Techniques/standards , Botulinum Toxins/analysis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Limit of Detection , Luminescence , Phosphoproteins/immunology , Proto-Oncogene Proteins c-bcl-2/analysis , Receptor, ErbB-2/analysis , Sensitivity and Specificity , Viral Matrix Proteins/immunology
14.
Acta Virol ; 64(4): 417-426, 2020.
Article in English | MEDLINE | ID: covidwho-914659

ABSTRACT

Aquatic birds are the main reservoir of influenza A viruses (IAVs). These viruses can infect humans repeatedly and cause acute respiratory disease with potential of spread in the form of epidemics. In addition, avian influenza viruses that overcome the interspecies barrier and adapt to humans can cause a world-wide pandemic with severe consequences to human health. Therefore, scientists are focused on the development of a "universal" vaccine with a broad protective efficacy, i.e. against different subtypes of influenza A viruses and not only against the currently co-circulating human epidemic strains. Nowadays, several new vaccine design strategies have been described. Most of them utilize the conserved stem part of influenza surface glycoprotein hemagglutinin (HA) or the ectodomain of M2 (M2e) protein with proton-channel activity. A comparison of the efficacy of novel vaccines and their protective mechanisms against influenza infection is discussed in this review and should be considered for the construction of the most effective broadly protective vaccine with minimal side effects. This is the essential goal in influenza virus research today, especially when the infection with new human coronavirus SARS-CoV-2 can interfere with the course of influenza virus infection. Keywords: influenza A virus; HA2 gp; M2 ectodomain; universal vaccine.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A virus , Influenza Vaccines/immunology , Influenza, Human , Viral Matrix Proteins/immunology , Animals , COVID-19 , Humans , Influenza A virus/genetics , Influenza, Human/prevention & control , Orthomyxoviridae Infections , SARS-CoV-2
15.
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
16.
Cell Mol Immunol ; 18(3): 613-620, 2021 03.
Article in English | MEDLINE | ID: covidwho-894385

ABSTRACT

A novel SARS-related coronavirus (SARS-CoV-2) has recently emerged as a serious pathogen that causes high morbidity and substantial mortality. However, the mechanisms by which SARS-CoV-2 evades host immunity remain poorly understood. Here, we identified SARS-CoV-2 membrane glycoprotein M as a negative regulator of the innate immune response. We found that the M protein interacted with the central adaptor protein MAVS in the innate immune response pathways. This interaction impaired MAVS aggregation and its recruitment of downstream TRAF3, TBK1, and IRF3, leading to attenuation of the innate antiviral response. Our findings reveal a mechanism by which SARS-CoV-2 evades the innate immune response and suggest that the M protein of SARS-CoV-2 is a potential target for the development of SARS-CoV-2 interventions.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , COVID-19/immunology , Immunity, Innate , SARS-CoV-2/immunology , Signal Transduction/immunology , Viral Matrix Proteins/immunology , HEK293 Cells , HeLa Cells , Humans
17.
PLoS One ; 15(10): e0240577, 2020.
Article in English | MEDLINE | ID: covidwho-874195

ABSTRACT

The causative agent of severe acute respiratory syndrome (SARS) reported by the Chinese Center for Disease Control (China CDC) has been identified as a novel Betacoronavirus (SARS-CoV-2). A computational approach was adopted to identify multiepitope vaccine candidates against SARS-CoV-2 based on S, N and M proteins being able to elicit both humoral and cellular immune responses. In this study, the sequence of the virus was obtained from NCBI database and analyzed with in silico tools such as NetMHCpan, IEDB, BepiPred, NetCTL, Tap transport/proteasomal cleavage, Pa3P, GalexyPepDock, I-TASSER, Ellipro and ClusPro. To identify the most immunodominant regions, after analysis of population coverage and epitope conservancy, we proposed three different constructs based on linear B-cell, CTL and HTL epitopes. The 3D structure of constructs was assessed to find discontinuous B-cell epitopes. Among CTL predicted epitopes, S257-265, S603-611 and S360-368, and among HTL predicted epitopes, N167-181, S313-330 and S1110-1126 had better MHC binding rank. We found one putative CTL epitope, S360-368 related to receptor-binding domain (RBD) region for S protein. The predicted epitopes were non-allergen and showed a high quality of proteasomal cleavage and Tap transport efficiency and 100% conservancy within four different clades of SARS-CoV-2. For CTL and HTL epitopes, the highest population coverage of the world's population was calculated for S27-37 with 86.27% and for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%, respectively. We identified overall 10 discontinuous B-cell epitopes for three multiepitope constructs. All three constructs showed strong interactions with TLRs 2, 3 and 4 supporting the hypothesis of SARS-CoV-2 susceptibility to TLRs 2, 3 and 4 like other Coronaviridae families. These data demonstrated that the novel designed multiepitope constructs can contribute to develop SARS-CoV-2 peptide vaccine candidates. The in vivo studies are underway using several vaccination strategies.


Subject(s)
Coronavirus Infections/prevention & control , Epitopes/immunology , Nucleocapsid Proteins/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/immunology , Viral Matrix Proteins/immunology , COVID-19 , Coronavirus Infections/immunology , Coronavirus Nucleocapsid Proteins , Epitopes/chemistry , HLA Antigens/chemistry , HLA Antigens/immunology , Humans , Molecular Docking Simulation , Nucleocapsid Proteins/chemistry , Phosphoproteins , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/chemistry , Toll-Like Receptors/chemistry , Toll-Like Receptors/metabolism , Vaccines, Subunit/chemistry , Viral Matrix Proteins/chemistry
18.
Sci Rep ; 10(1): 16219, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-811544

ABSTRACT

COVID-19 pandemic has resulted in 16,114,449 cases with 646,641 deaths from the 217 countries, or territories as on July 27th 2020. Due to multifaceted issues and challenges in the implementation of the safety and preventive measures, inconsistent coordination between societies-governments and most importantly lack of specific vaccine to SARS-CoV-2, the spread of the virus that initially emerged at Wuhan is still uprising after taking a heavy toll on human life. In the present study, we mapped immunogenic epitopes present on the four structural proteins of SARS-CoV-2 and we designed a multi-epitope peptide based vaccine that, demonstrated a high immunogenic response with a vast application on world's human population. On codon optimization and in-silico cloning, we found that candidate vaccine showed high expression in E. coli and immune simulation resulted in inducing a high level of both B-cell and T-cell mediated immunity. The results predicted that exposure of vaccine by administrating three injections significantly subsidized the antigen growth in the system. The proposed candidate vaccine found promising by yielding desired results and hence, should be validated by practical experimentations for its functioning and efficacy to neutralize SARS-CoV-2.


Subject(s)
Epitopes/immunology , Molecular Docking Simulation , Vaccines, Subunit/immunology , Viral Vaccines/immunology , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Antigens, Viral/immunology , B-Lymphocytes/immunology , COVID-19 Vaccines , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Nucleocapsid Proteins , Epitopes/chemistry , HLA Antigens/chemistry , HLA Antigens/immunology , Humans , Immunogenicity, Vaccine , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/immunology , Phosphoproteins , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Toll-Like Receptors/immunology , Vaccines, Subunit/chemistry , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/immunology , Viral Vaccines/chemistry
19.
Cell Mol Immunol ; 17(10): 1098-1100, 2020 10.
Article in English | MEDLINE | ID: covidwho-772968
20.
Infect Dis Poverty ; 9(1): 132, 2020 Sep 16.
Article in English | MEDLINE | ID: covidwho-768657

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) linked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause severe illness and life-threatening pneumonia in humans. The current COVID-19 pandemic demands an effective vaccine to acquire protection against the infection. Therefore, the present study was aimed to design a multiepitope-based subunit vaccine (MESV) against COVID-19. METHODS: Structural proteins (Surface glycoprotein, Envelope protein, and Membrane glycoprotein) of SARS-CoV-2 are responsible for its prime functions. Sequences of proteins were downloaded from GenBank and several immunoinformatics coupled with computational approaches were employed to forecast B- and T- cell epitopes from the SARS-CoV-2 highly antigenic structural proteins to design an effective MESV. RESULTS: Predicted epitopes suggested high antigenicity, conserveness, substantial interactions with the human leukocyte antigen (HLA) binding alleles, and collective global population coverage of 88.40%. Taken together, 276 amino acids long MESV was designed by connecting 3 cytotoxic T lymphocytes (CTL), 6 helper T lymphocyte (HTL) and 4 B-cell epitopes with suitable adjuvant and linkers. The MESV construct was non-allergenic, stable, and highly antigenic. Molecular docking showed a stable and high binding affinity of MESV with human pathogenic toll-like receptors-3 (TLR3). Furthermore, in silico immune simulation revealed significant immunogenic response of MESV. Finally, MEV codons were optimized for its in silico cloning into the Escherichia coli K-12 system, to ensure its increased expression. CONCLUSION: The MESV developed in this study is capable of generating immune response against COVID-19. Therefore, if designed MESV further investigated experimentally, it would be an effective vaccine candidate against SARS-CoV-2 to control and prevent COVID-19.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Humans , Immunogenicity, Vaccine/immunology , Molecular Docking Simulation , Pneumonia, Viral/immunology , SARS-CoV-2 , Sequence Analysis, Protein , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/immunology , Vaccines, Subunit/chemistry , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccinology/methods , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Vaccines/chemistry , Viral Vaccines/genetics
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