Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
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.
J Infect Dis ; 224(8): 1305-1315, 2021 10 28.
Article in English | MEDLINE | ID: covidwho-1493821

ABSTRACT

BACKGROUND: A notable feature of coronavirus disease 2019 (COVID-19) is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. METHODS: We investigated the presence of cross-reactive anti-SARS-CoV-2 IgG antibodies to spike 1 (S1), S1-receptor-binding domain (S1-RBD), and nucleocapsid protein (NP) by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralization assay, in prepandemic sera collected from children (n = 50) and adults (n = 45), and compared with serum samples from convalescent COVID-19 patients (n = 16). RESULTS: A significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD, and NP antigens, and the anti-S1 and anti-S1-RBD antibody levels correlated with anti-HCoV-HKU1 and anti-HCoV-OC43 S1 antibody titers in prepandemic samples (P < .001). There were marked increases of anti-HCoV-HKU1 and - OC43 S1 (but not anti-NL63 and -229E S1-RBD) antibody titers in serum samples from convalescent COVID-19 patients (P < .001), indicating an activation of cross-reactive immunological memory to ß-coronavirus spike. CONCLUSIONS: We demonstrated cross-reactive anti-SARS-CoV-2 antibodies in prepandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Immunoglobulin G/blood , SARS-CoV-2/immunology , Adolescent , Adult , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , Child , Child, Preschool , Convalescence , Coronavirus 229E, Human/immunology , Coronavirus Envelope Proteins/immunology , Coronavirus OC43, Human/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , Humans , Immunoglobulin G/immunology , Immunologic Memory , Male , Middle Aged , Spike Glycoprotein, Coronavirus/immunology , Young Adult
3.
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
4.
Front Immunol ; 12: 724060, 2021.
Article in English | MEDLINE | ID: covidwho-1430701

ABSTRACT

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRß1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world's population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides' potential coverage for the world populations up to 62.9%. These peptides' 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology's potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , Coronavirus Envelope Proteins/immunology , Coronavirus M Proteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Aotidae , COVID-19/prevention & control , HLA-DRB1 Chains/genetics , Humans , Peptides/immunology , SARS-CoV-2/immunology
5.
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
6.
Int J Mol Sci ; 22(16)2021 Aug 23.
Article in English | MEDLINE | ID: covidwho-1367852

ABSTRACT

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


Subject(s)
Antibodies, Viral/metabolism , COVID-19/immunology , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/pathogenicity , Viral Matrix Proteins/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , Cell Line , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/immunology , Coronavirus Envelope Proteins/ultrastructure , Cricetinae , Humans , Microscopy, Electron, Transmission , Mutation , Neutralization Tests , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Matrix Proteins/ultrastructure , Virion/genetics , Virion/immunology , Virion/metabolism , Virion/ultrastructure
7.
mBio ; 12(3): e0122921, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1286719

ABSTRACT

We sought to discover links between antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and patient clinical variables, cytokine profiles, and antibodies to endemic coronaviruses. Serum samples from 30 patients of younger (26 to 39 years) and older (69 to 83 years) age groups and with varying clinical severities ranging from outpatient to mechanically ventilated were collected and used to probe a novel multi-coronavirus protein microarray. This microarray contained variable-length overlapping fragments of SARS-CoV-2 spike (S), envelope (E), membrane (M), nucleocapsid (N), and open reading frame (ORF) proteins created through in vitro transcription and translation (IVTT). The array also contained SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus OC43 (HCoV-OC43), and HCoV-NL63 proteins. IgG antibody responses to specific epitopes within the S1 protein region spanning amino acids (aa) 500 to 650 and within the N protein region spanning aa 201 to 300 were found to be significantly higher in older patients and further significantly elevated in those older patients who were ventilated. Additionally, there was a noticeable overlap between antigenic regions and known mutation locations in selected emerging SARS-CoV-2 variants of current clinical consequence (B.1.1.7, B1.351, P.1, CAL20.C, and B.1.526). Moreover, the older age group displayed more consistent correlations of antibody reactivity with systemic cytokine and chemokine responses than the younger adult group. A subset of patients, however, had little or no response to SARS-CoV-2 antigens and disproportionately severe clinical outcomes. Further characterization of these slow-low-responding individuals with cytokine analysis revealed significantly higher interleukin-10 (IL-10), IL-15, and interferon gamma-induced protein 10 (IP-10) levels and lower epidermal growth factor (EGF) and soluble CD40 ligand (sCD40L) levels than those of seroreactive patients in the cohort. IMPORTANCE As numerous viral variants continue to emerge in the coronavirus disease 2019 (COVID-19) pandemic, determining antibody reactivity to SARS-CoV-2 epitopes becomes essential in discerning changes in the immune response to infection over time. This study enabled us to identify specific areas of antigenicity within the SARS-CoV-2 proteome, allowing us to detect correlations of epitopes with clinical metadata and immunological signals to gain holistic insight into SARS-CoV-2 infection. This work also emphasized the risk of mutation accumulation in viral variants and the potential for evasion of the adaptive immune responses in the event of reinfection. We additionally highlighted the correlation of antigenicity between structural proteins of SARS-CoV-2 and endemic HCoVs, raising the possibility of cross-protection between homologous lineages. Finally, we identified a subset of patients with minimal antibody reactivity to SARS-CoV-2 infection, prompting discussion of the potential consequences of this alternative immune response.


Subject(s)
Antibodies, Viral/blood , Coronavirus NL63, Human/immunology , Coronavirus OC43, Human/immunology , Cytokines/blood , Middle East Respiratory Syndrome Coronavirus/immunology , SARS-CoV-2/immunology , Adult , Age Factors , Aged , Aged, 80 and over , Antibodies, Viral/immunology , COVID-19/immunology , Coronavirus Envelope Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoglobulin G/immunology , Male , Phosphoproteins/immunology , Protein Array Analysis , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology
8.
Clin Immunol ; 227: 108733, 2021 06.
Article in English | MEDLINE | ID: covidwho-1198654

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for many pathological processes, including altered vascular disease development, dysfunctional thrombosis and a heightened inflammatory response. However, there is limited work to determine the underlying cellular responses induced by exposure to SARS-CoV-2 structural proteins. Thus, our objective was to investigate how human arterial adventitial fibroblasts inflammation, thrombosis and diabetic disease markers are altered in response to Spike, Nucleocapsid and Membrane-Envelope proteins. We hypothesized that after a short-term exposure to SARS-CoV-2 proteins, adventitial fibroblasts would have a higher expression of inflammatory, thrombotic and diabetic proteins, which would support a mechanism for altered vascular disease progression. After incubation, the expression of gC1qR, ICAM-1, tissue factor, RAGE and GLUT-4 was significantly up-regulated. In general, the extent of expression was different for each SARS-CoV-2 protein, suggesting that SARS-CoV-2 proteins interact with cells through different mechanisms. Thus, SARS-CoV-2 protein interaction with vascular cells may regulate vascular disease responses.


Subject(s)
COVID-19/immunology , Cardiovascular Diseases/virology , Diabetes Mellitus/virology , Fibroblasts/metabolism , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Thrombosis/virology , Aorta/cytology , Aorta/metabolism , Cardiovascular Diseases/complications , Cardiovascular Diseases/immunology , Cardiovascular Diseases/metabolism , Carrier Proteins/metabolism , Cell Survival/immunology , Cell Survival/physiology , Complement System Proteins/immunology , Coronavirus Envelope Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Diabetes Mellitus/metabolism , Glucose Transporter Type 4/metabolism , Humans , Inflammation/metabolism , Inflammation/virology , Intercellular Adhesion Molecule-1/metabolism , Mitochondrial Proteins/metabolism , Receptor for Advanced Glycation End Products/metabolism , Thrombosis/complications , Thrombosis/metabolism
9.
J Infect Dis ; 223(9): 1555-1563, 2021 05 20.
Article in English | MEDLINE | ID: covidwho-1069262

ABSTRACT

Repeated infections with endemic human coronaviruses (hCoV) are thought to reflect lack of long-lasting protective immunity. We evaluated circulating human CD4 T cells collected prior to 2020 for reactivity towards hCoV spike proteins, probing for the ability to produce interferon-γ, interleukin-2, or granzyme B. We found robust reactivity to spike-derived epitopes, comparable to influenza, but highly variable abundance and functional potential across subjects, depending on age and viral antigen specificity. To explore potential of these memory cells to be recruited in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we examined the subjects for cross-reactive recognition of epitopes from SARS-CoV-2 nucleocapsid, membrane/envelope, and spike. Functional potential of these cross-reactive CD4 T cells was highly variable; nucleocapsid-specific CD4 T cells but not spike-reactive cells showed exceptionally high levels of granzyme production upon stimulation. These results are considered in light of recruitment of hCoV-reactive cells into responses to SARS-CoV infections or vaccinations.


Subject(s)
CD4-Positive T-Lymphocytes/virology , COVID-19/immunology , Coronavirus Infections/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Adult , Aged , Coronavirus Envelope Proteins/immunology , Coronavirus M Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Cross Reactions , Granzymes/metabolism , Humans , Immunologic Memory , Interferon-gamma/metabolism , Interleukin-2/metabolism , Middle Aged , Spike Glycoprotein, Coronavirus/immunology
10.
Int J Biol Macromol ; 172: 74-81, 2021 Mar 01.
Article in English | MEDLINE | ID: covidwho-1002618

ABSTRACT

COVID-19 is one of the fatal pandemic throughout the world. For cellular fusion, its antigenic peptides are presented by major histocompatibility complex (MHC) in humans. Therefore, exploration into residual interaction details of CoV2 with MHCs shall be a promising point for instigating the vaccine development. Envelope (E) protein, the smallest outer surface protein from SARS-CoV2 genome was found to possess the highest antigenicity and is therefore used to identify B-cell and T-cell epitopes. Four novel mutations (T55S, V56F, E69R and G70del) were observed in E-protein of SARS-CoV2 after evolutionary analysis. It showed a coil➔helix transition in the protein conformation. Antigenic variability of the epitopes was also checked to explore the novel mutations in the epitope region. It was found that the interactions were more when SARS-CoV2 E-protein interacted with MHC-I than with MHC-II through several ionic and H-bonds. Tyr42 and Tyr57 played a predominant role upon interaction with MHC-I. The higher ΔG values with lesser dissociation constant values also affirm the stronger and spontaneous interaction by SARS-CoV2 proteins with MHCs. On comparison with the consensus E-protein, SARS-CoV2 E-protein showed stronger interaction with the MHCs with lesser solvent accessibility. E-protein can therefore be targeted as a potential vaccine target against SARS-CoV2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , Coronavirus Envelope Proteins/immunology , Evolution, Molecular , Molecular Docking Simulation , SARS-CoV-2/immunology , Amino Acid Sequence , Coronavirus Envelope Proteins/chemistry , Coronavirus Envelope Proteins/genetics , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Humans , Hydrogen Bonding , Kinetics , Mutation/genetics , Phylogeny , Protein Binding , Solvents , Thermodynamics , Viral Vaccines/immunology
11.
Virus Res ; 291: 198222, 2021 01 02.
Article in English | MEDLINE | ID: covidwho-912664

ABSTRACT

The envelope glycoprotein (E) is the smallest structural component of SARS-CoVs; plays an essential role in the viral replication starting from envelope formation to assembly. The in silico analysis of 2086 whole genome sequences from India performed in this study provides the first observation on the extensive deletion of amino acid residues in the C-terminal region of the envelope glycoprotein in 34 Indian SARS-CoV-2 genomes. These amino acid deletions map to the homopentameric interface and PDZ binding motif (PBM) present in the C-terminal region of E protein as well as immediately after the reverse primer binding region as per Charité protocol in 26 of these genomes, hence, their detection through RT-qPCR may not be hampered and therefore E gene-based RT-qPCR would still detect these isolates. Eight genomes from the State of Odisha had deletion even in the primer binding site. It is possible that the deletions in the C-terminal region of E protein of these genomes are a result of adapting to a newer geographical area and host. The information on the clinical status was available only for 9 out of 34 cases and these were asymptomatic. However, further studies are indispensable to understand the functional consequences of amino acid deletion in the C terminal region of SARS-CoV-2 envelope protein in the viral pathogenesis and host adaptation.


Subject(s)
Coronavirus Envelope Proteins/genetics , SARS-CoV-2/genetics , Adult , Amino Acid Sequence , Computer Simulation , Coronavirus Envelope Proteins/immunology , Epitopes, B-Lymphocyte , Female , Gene Deletion , Genome, Viral , Humans , India , Male , Middle Aged , Real-Time Polymerase Chain Reaction , SARS-CoV-2/isolation & purification
SELECTION OF CITATIONS
SEARCH DETAIL
...