Learn More. The spike protein of coronavirus is key target for drug development and other pharmacological interventions.
In current study, we performed an integrative approach to predict antigenic sites in SARS-CoV-2 spike receptor binding domain and found nine potential antigenic sites. The predicted antigenic sites were then assessed for possible molecular similarity with other known antigens in different organisms.
Out of nine sites, seven sites showed molecular similarity with 54 antigenic determinants found in twelve pathogenic bacterial species Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Borrelia burgdorferi, Clostridium perfringens, Clostridium tetani, Helicobacter Pylori, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholera and Yersinia pestis , two malarial parasites Plasmodium falciparum and Plasmodium knowlesi and influenza virus A.
Antigens from Mycobacterium that showed similarity were mainly involved in modulating host cell immune response and ensuring persistence and survival of pathogen in host cells.
Presence of a large number of antigenic determinants, similar to those in highly pathogenic microorganisms, not merely accounts for complex etiology of the disease but also provides an explanation for observed pathophysiological complications, such as deregulated immune response, unleashed or dysregulated cytokine secretion cytokine storm , multiple organ failure etc.
Over-representation of antigenic determinants from Plasmodium and Mycobacterium in all antigenic sites suggests that anti-malarial and anti-TB drugs can prove to be clinical beneficial for COVID treatment. All epitopes possessed significantly lower predicted IC50 value which is a prerequisite for a preferred vaccine candidate for COVID The recent outbreak of novel coronavirus SARS-CoV-2 earlier known as nCoV in Wuhan city of China has led to serious health crisis as well as impacted socio-economic development globally.
The ability of coronaviruses to enter into host cells and infect them is due to their ability to establish strong attachment with host cell receptor proteins such as integrins and others via their RGD motif present in their receptor binding domain of spike glycoproteins Dakal, , Li et al. The RGD motifs are well known in the field of cell biology, cell therapy and tissue engineering because of their remarkable cell-adhesive property Bellis, Extracellular matrix proteins, such as fibronectin and laminin, possess RGD motif and are frequently coated onto the surface of biomaterials petri-dishes, T-flasks for facilitating human cells adhesion onto the surface of the biomaterials Bellis, Initially the spike proteins bind to cellular receptor angiotensin-converting enzyme 2 ACE2.
Following the cleavage of spike protein and with the help of proteases produce 2 domains of spike protein, S1 known as the receptor binding domain, which recognizes and bind to the RBD and S2 which fuse with the membrane following the entry of the virus into the host cell Hoffmann et al.
After that the viral RNA get exposed in the cytoplasm of the host cell. Virus infection in humans results in two major types of immune response. The first is an innate immune response, which is first line of defense and involves synthesis of proteins called interferons key regulators of viral replication and stimulation of a number of immune cells Theofilopoulos et al.
After recognizing pathogenic features in the invading molecule, virus is delivered to macrophage and dendritic cells DCs that play crucial roles for viral destruction and immune response induction in mucosal-associated lymphoid tissues MALT Spiegel et al. On activation, an unleashed production of inflammatory cytokines and chemokines can be seen in the tissues and serum of the COVID patients, a pathophysiological condition formally known as cytokine storm Mehta et al.
In cases when the innate response is not enough to prevent viral infection, adaptive immunity comes into action, especially during the later stages of viral infection in which infection has already proceeded beyond the first few rounds of viral replication. As the part of adaptive immune response, antigen presenting cells such as B-cells, macrophages and dendritic cells DCs recognize and present viral antigens and trigger activation and proliferation of T-helper Th cells.
The T-helper cells are required for the generation of the humoral response the synthesis of virus-specific antibodies by B lymphocytes and the cell-mediated response recognition and targeted killing of virus infected antigen-displaying altered self-cells by cytotoxic T-cells.
The respiratory dendritic cells rDCs that resides in lung epithelium acquire the invading virus or its antigens from infected lung cells and become activated Peebles and Graham, , Tognarelli et al. Under the influence of IL-2, IL and other cytokines and chemokines, natural killer NK cells also develop and target virus-infected cells using antibody dependent cell-mediated cytotoxicity ADCC Cooper et al. These effector molecules cytokines, chemokines and cytotoxic molecules in a multi-faceted way inhibit viral replication and enhance antigen presentation leading to recruitment of additional immune cells such as NK-cells of innate and adaptive system at the site of viral infection for destroying infected epithelial cells virus-infected altered self-cells and eliminating virus Cerwenka et al.
In particular, the receptor binding domain RBD , which stretches from residue to within the spike protein, is the most important structural module Dakal, , Wong et al. Several researchers demonstrated that spike protein of SARS-CoV-1 plays a key role in eliciting potent T-cell responses and binds with neutralizing-antibodies Prabakaran et al. In particular, the S1 glycoprotein of SARS-CoV-1spike protein has been found be an important immunodominant epitope which induces a number of neutralizing antibodies Tian et al.
Of all structural proteins in SARS-CoV-2, spike proteins are the first to interact with the host cells, and therefore, the initial host cell immune response in COVID patients is expected to be against the exposed antigenic epitopes in the spike proteins. This suggested that there is a strong need for exploration and prediction of antigenic sites and potent cytotoxic T-cell epitopes in spike RBD for rapid developments of novel vaccine candidates and neutralizing antibodies against SARS-CoV-2 infection.
MHC molecules, also called as human leukocyte-associated HLA antigens, are cell surface glycoproteins that bind peptide fragments of proteins that either have been synthesized within the cell class I MHC molecules or that have been ingested by the cell and proteolytically processed class II MHC molecules.
The structural modeling was done using Chimera ver. The antigenic propensity was predicted using Antigenic Peptide tool of Universidad Complutense Madrid imed. The tool employs support vector machine SVM in which the tripeptide similarity score and the propensity scores are combined to yield improved predictions Yao et al.
The antigenic sites were used as input in tripeptide format and output was obtained in the form of comprehensive information about experimentally verified epitopes from a wide range of known antigens. The prediction is based on known antigens that have been shown to be presented by MHC class I proteins in various experimental studies and are capable of inducing potent CTL immune responses.
Alleles of this HLA class are normally express on professional antigen-presenting cells APCs such as B lymphocytes, dendritic cells, mononuclear phagocytes, endothelial cells and thymic epithelial cells that are crucial in exacerbating immune responses in multi-faceted manner. The length of these antigenic sites stretched from four amino acids to twenty three amino acids.
We performed structural analysis of spike RBD domain using Chimera and found that all the predicted antigenic sites are surface exposed loops of the spike RBD validating the ElliPro prediction Fig. All predicted sites are marked with their sequence and position. The predicted antigenic sites were then submitted to AntigenDB database for assessing their molecular similarity with other known antigens or antigenic determinants in different microorganisms.
While using the whole antigenic site, we could not predict any similar antigenic determinant and as such the antigenic sites predicted in SARS-CoV-2 spike RBD appeared to be unique. To our surprise, the predicted antigenic sites showed similarity with antigenic determinants from twelve pathogenic bacterial species Mycobacterium tuberculosis, Mycobacterium leprae, Bacillus anthracis, Borrelia burgdorferi, Clostridium perfringens, Clostridium tetani, Helicobacter Pylori, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholera and Yersinia pestis , two malaria parasites Plasmodium falciparum and Plasmodium knowlesi and Influenza virus A.
All these microorganisms are well known for their pathogenesis and are associated with life threatening diseases in humans and in some animals as well.
In total, out of nine sites, seven sites showed molecular similarity with 54 antigens from twelve pathogenic bacterial species, two malarial parasites and influenza virus A. The predicted molecular similarity between antigenic sites in SARS-CoV-2 and antigenic determinants from pathogenic organisms has three major implications: 1 the human immune system will recognize and activate the immune cells and trigger response in response to SARS-CoV-2 infection in the similar manner as the antigenic determinants could arouse after infection by these fifteen pathogenic organisms, 2 the pathophysiological outcomes and clinical features symptoms observed in COVID patients are expected to be similar to the symptoms in any patient infected by any of these fifteen microorganisms Huang et al.
Antigenic sites in SARS-CoV-2 showed similarity with a number of proteins, enzymes, toxins and virulent factors with diverse functional role in pathogens. The role of proteins, enzymes, toxins and virulence factors has been presented here. Diacylglycerolacyltransferase fbpB from M. Tuberculosis have high affinity for extracellular matrix protein, fibronectin, which facilitates strong binding of M. The kDa chaperonin groS of M.
Leprae is implicated for its virulence Roberts et al. An in vitro study using recombinant M. The 6 kDa early secretory antigenic target of M. The lipoprotein Psts1 of M. Another lipoprotein Lpqh of M. The MPT64 mpt64 of M. Sequence of DNA polymerase 1 of M. In nutshell, the antigenic sites in SARS-CoV-2were similar to those found in pathogenic proteins in Mycobacterium having role in modulating the immune response for escaping immune recognition and ensuring persistence in host body, possibly in latent stage.
The spike RBD was also found to possess antigenic determinants as found in proteins from malarial parasites such as Plasmodium falciparum and Plasmodium knowlesi. All these proteins were found to be present in the sera of patients exposed natural to sporozoite or were immunized with it Doolan et al. Circumsporozoite proteins CSPs are important malarial sporozoite protein having role in liver cells invasion in humans Doolan et al.
The RESA migrates to the host cell membrane, where it binds to spectrin; however, the mechanism and type of binding and its pathological consequences are largely unknown yet Pei et al. The proteins are hemagglutinin HA one of the three transmembrane protein of virus and nucleoprotein NP encapsulated in viral nucleocapsids.
While HA is involved in viral assembly at host cell membrane, the viral nucleoproteins helps in incorporation of viral genetic material into newly budded virions Zhang et al.
The nucleoproteins of influenza virus have RNA binding and protein interaction sites that may be important for their host cell functions such as stability and nuclear export of mRNA Krug, , Qian et al. Two antigenic determinants predicted from Bacillus anthracis were of protective antigen pagA and lethal factor. Five antigenic determinants predicted from spirochete Borrelia burgdorferi were of flagellar filament core protein Fla1 and outer surface proteins A OspA and C OspC. Fla1 protein is an immunodominant41kDaantigen present in the sera of Lyme-disease patients that allows bacteria to efficiently bore into the host cells for colonization and survival using outer surface proteins Steere et al.
The antigenic sequences predicted from Clostridium perfringens and Clostridium tetani were also of the toxin proteins such as heat-labile enterotoxin B chain and tetanus toxin. Similarly, some other predicted antigenic determinants were also of well known toxins and virulent factor such as enterotoxin A from Staphylococcus aureus , M protein from Streptococcus pyogenes , cholera enterotoxin subunit B and toxin coregulated pilin from Vibrio cholerae.
The30kDa urease subunit alpha ureA from Helicobacter pylori acts as virulence factor and cause infection in stomach via host-pathogen interaction Schoep et al. The endopeptidase p60 Lm-p60 of Listeria monocytogenes whose sequence was also predicted in similar search is a highly conserved carbohydrate binding module which can be engineered for binding to peptidoglycans with high affinity Yu et al. These play role in bacterial virulence and act as chaperone to prevent membrane lysis in M.
F1 capsule of Yersinia pestis helps bacteria avoid up taken by macrophages Levy et al. In total fourteen peptide sequences were recognized as possible epitopes for T-cells predicted against MHC class I and only eight peptide sequences were predicted to be overlapping with the antigenic sites predicted Table 4.
However, the predicted IC50 value for six peptides was very high and thus these peptides failed to qualify the criteria of becoming a potent vaccine candidate. Alleles of this HLA class are normally found on professional antigen-presenting cells APCs such as B lymphocytes, dendritic cells, mononuclear phagocytes, endothelial cells and thymic epithelial cells. These cells are important in exacerbating immune responses in different ways.
Molecular similarity can be defined as the theoretical explanation for sequence similarities mainly in antigens between two or more organisms. The concept of molecular or antigen similarity has been well presented and entrusted in literature in context to autoimmune disorders Fujinami et al.
In context to current manuscript, the molecular similarity has been observed between antigenic sites in SARS-CoV-2 spike protein and similar antigenic determinants in fifteen microorganisms, including bacteria, parasites and viruses.
We believe that the presence of tripeptide sequence s displaying molecular similarity with highly potent antigenic determinants as present in fifteen pathogenic microorganisms in the antigenic sites of SARS-CoV-2 spike proteins are sufficient to activate host immune cells T or B cells or other immune cells in similar as the fifteen microorganisms can do. Additionally, other researchers demonstrated how an antigenic tripeptide motif in the amino acid sequence of a protein in pathogens can also differentially activate one of the two immune cells, either B-cell or T-cell Yao et al.
The molecular similarity of spike RBD with antigenic determinants as found in different pathogenic microorganisms is expected to induce exuberant innate and adaptive immune responses leading to excessive secretion of cytokines that is expected to adversely affect several vital organs leading to multi-organ failure in COVID patients Fig.
Since, T-cell play critical role in controlling immune response and one can expect deregulated immune response and dysregulated cytokine secretion in aged and immune-compromised patients who have less number of T-cells in their body Kim et al.
There is also a correlation between the secretion of some cytokines and chemokines, such as IL-6, IL-8, and MCP-1 and IP, with higher mortality and severity of disease Reghunathan et al. The study explains the complex etiology and pathophysiology of COVID and sheds light on implications and future prospects. Presence of antigenic sites in SARS-CoV-2 with similarity with antigenic determinants found in pathogenic bacterial, malarial and viral species is seriously alarming as their presence makes the SARS-CoV-2 more pathogenic than any other previously known coronavirus.
The antigenic patches from pathogenic microorganisms in SARS-CoV-2 could be traced only as small tripeptide motifs and this makes their origin uncertain.
However, most of the antigenic determinants found in pathogenic microorganisms were related to antigenic proteins having established function as toxin and proven role in virulence and pathogenicity.
Besides this, other antigenic determinants found similar to Mycobacterium antigens having role in persistence and survival of pathogen in host cells. Some antigenic determinants from HA and NP of influenza virus A were also in antigenic sites of SARS-CoV-2 and these antigens from influenza virus have role in assembly of newly budded virions in case of influenza virus A. Due to the presence of antigenic sites with molecular similarity with antigens from different pathogenic organisms, parasites and virus, it can be ruled out that COVID patients may possibly suffer multi-organ failure as these pathogenic antigens are well known to cause serious damages to different vital organs of human body such as liver, kidney, blood, stomach, heart and bones.
This is also clearly evident from complex etiology and pathophysiological outcomes clinical features or symptoms as observed in COVID patients. The possible explanation is that the memory B or T cells previously generated by the microorganisms in context here would get activated again upon SARS-CoV-2 infection long-lived immunity to reinfection because of the similar antigenic specificity and due to presence of common antigenic determinants in both.
As such, nothing can be explicitly stated regarding the presence of antigenic determinants as found in this current study. We speculate that viruses may be doing this as a genetic trick for tackling genetic constraints imposed by host cells to ensure their persistence and survival.
The SARS-CoV-2 might have changed or switched the molecular composition of its spike protein using a process called as antigenic variation Smith, , Chibo and Birch, , de Jong et al. Antigenic variations are genetic tricks using which some viruses, such as influenza virus and coronavirus etc. Antigenic variations are brought up by periodic and random genetic mutations, generally in viral surface antigens, so as to temporarily camouflage host immune cells and to prevent clearance Smith, , de Jong et al.
The antigenic alteration can occur in two different ways: 1 genetic drift that causes subtle changes in amino acids, and 2 antigenic shift that causes major alterations in the antigenic properties of the protein Smith, , de Jong et al. Such antigenic-shifted strains of virus generate periodically when genes encoding structural proteins are acquired from viruses that infect animal hosts.
These antigenic-shifted strains are known to cause global pandemic and recurring acute infections Smith, , de Jong et al. Antigenic properties of spike glycoprotein of SARS-CoV-2, especially of the receptor binding domain RBD , were well appreciated by many experimental researchers including myself in current study Baruah and Bose, , Lucchese, Most of the vaccines such as virus vector and protein subunit vaccine developed or under clinical trails have been developed using full length spike protein or RBD of spike protein of SARS-CoV The identified sequence in current study are part of all vaccine under clinical or pre-clinical development.
Baruah and Bose, This study provides the first evidence in favor of antigenic variations in spike RBD of SARS-CoV-2 that succour virus to undergo adaptive evolution in order to infect humans for their survival and persistence. These antigenic variations not only explain the complex immunological aspects, etiology and pathophysiology of the disease but also suggest different therapeutics anti-malarial, anti-TB, anti-leprosy, anti-plague, anti-lyme, anti-anthrax, anti-cholera etc.
We envisage that several lines of research endeavors are still required towards understanding the multifaceted mechanisms of immunomodulation, host-virus interaction and SARS-CoV-2 infection.
TCD conceived the idea and designed the experimental strategy and designed methodology, performed the analyses, wrote the manuscript, revised the manuscript. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
National Center for Biotechnology Information , U. Prediction of antigenic epitopes is useful for the investigation to the mechanism in body self-protection systems and help during the design of vaccine components and immuno-diagnostic reagents.
Usually, B-cell antigenic epitopes are classified as either continuous or discontinuous. A continuous also called linear epitope is a consecutive fragment from the protein sequence, and a discontinuous epitope is composed of several fragments scattered along the protein sequence, which form the antigen-binding interface.
The boundary between continuous and discontinuous epitopes is vague; a fragment in a discontinuous epitope can be considered as a continuous epitope. Currently, the majority of available epitope prediction methods focus on continuous epitopes due to the convenience of the investigation in which the amino acid sequence of a protein is taken as the input.
Such prediction methods are based upon the amino acid properties including hydrophilicity , solvent accessibility , secondary structure , flexibility , and antigenicity.
In this work, we developed a new method to predict antigenic epitope with lastest sequence input from IEDB database.
Yao, L.
0コメント