Conserved SARS-CoV-2 Viral Peptides as Potential Prophylactic and Therapeutic Targets

Conserved SARS-CoV-2 Viral Peptides as Potential Prophylactic and Therapeutic Targets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Conserved SARS-CoV-2 Viral Peptides as Potential Prophylactic and Therapeutic Targets Radhakrishna Muttineni, Kalyani Putty, Jhansi Siripuram, Sreenidhi Ramamoorthy, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6798845/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The ongoing evolution of SARS-CoV-2, especially the emergence of heavily mutated variants like Omicron and its sub-lineages, has resulted in antigenic drift that diminishes the effectiveness of current first-generation vaccines, diagnostic tests, and treatments. This study employed a comprehensive immuno-informatics approach to identify highly conserved protein sequences from SARS-CoV-2 isolates reported in India. 1,33,154 complete protein sequences retrieved from the GISAID database between September 2021 and March 2023 were analysed. Results: The analysis revealed a total of 62,94,995 mutations, which include 66,861 unique mutations. Sequences comprising at least eight consecutive amino acids with mutation frequencies below 0.1% were considered conserved regions. This analysis identified 270 conserved sequences across both structural and non-structural proteins. Of these, 73 sequences were found to be antigenic and non-allergenic and were mapped onto their respective crystal structure of proteins to evaluate their functional relevance. Many conserved sequences overlapped with the known functionally significant epitopes conserved across SARS-CoV-2 variants, underscoring their importance. Conclusions: The identified conserved sequences offer valuable targets for developing variant-resilient peptide-based diagnostics, monoclonal antibody therapeutics, and multi-epitope peptide vaccines. This study provides a curated collection of conserved SARS-CoV-2 protein regions identified from Indian clinical isolates and emphasises their potential for diagnostic and therapeutic applications. These findings may contribute to developing universal, variant-proof strategies for SARS-CoV-2 detection, prevention, and treatment. SARS-CoV-2 COVID-19 Structural proteins Non-structural proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Background ​ The rapid emergence of SARS-CoV-2 variants has challenged the efficacy of first-generation vaccines. For instance, the heavily mutated Omicron variant evades many neutralising antibodies from prior infection or vaccination (Choi et al., 2022). This immune escape has led to breakthrough infections and prompted the rollout of variant-updated booster vaccines (e.g. bivalent BA.4/5 and monovalent XBB.1.5 boosters) to restore protection (Muik et al., 2024). Emerging COVID-19 vaccines increasingly focus on conserved viral epitopes to achieve broader protection. Strategies include multi-epitope vaccines incorporating conserved B- and T-cell epitopes across the SARS-CoV-2 proteome(C. Y. Wang et al., 2023a) The SARS-CoV-2 genome is approximately 30,000 nucleotides long and encodes 29 proteins, with four structural proteins, 16 non-structural proteins (NSPs), and nine accessory proteins (Bai et al., 2021). Recent studies revealed that including peptides from internal proteins like nucleocapsid (N) and membrane (M) alongside Spike (S) elicit robust CD4⁺ and CD8⁺ T-cell responses to complement neutralising antibodies. These peptides are also reported to induce potent and long-lasting B- and T-cell immunity in clinical trials, suggesting that they confer broad protection against diverse variants (Hsieh et al., 2024; Prakash et al., 2024a; Sankhala et al., 2024; C. Y. Wang et al., 2023b). T-cell-mediated immunity has proven more resilient to viral variation; studies show that most T-cell epitopes remain unchanged across variants (Prakash et al., 2024b). Notably, > 95% of CD8⁺ T-cell epitopes identified in the original strain are still conserved in Omicron (Choi et al., 2022). This preservation of conserved peptide targets helps maintain T-cell recognition and protection against severe disease despite the antigenic drift of SARS-CoV-2 (Choi et al., 2022). These reports underscore why next-generation vaccines are pivoting to include conserved elements to confer immunity that endures against current and future variants​ (Prakash et al., 2024b). Computational immunology has been instrumental in pinpointing conserved SARS-CoV-2 peptide targets. Immuno-informatics leverages the growing genomic data from SARS-CoV-2 variants and related coronaviruses to map regions of high sequence conservation. These conserved epitopes are often functionally important; mutation-intolerant regions are attractive targets for the broad-spectrum vaccine and serve as potential regions for the design of diagnostic and therapeutic approaches​. In silico tools also predict the binding of these epitopes to the common human MHC alleles, ensuring broad population coverage (Prakash et al., 2024b)​. By integrating sequence alignment, structural modelling, and epitope prediction algorithms, researchers can efficiently prioritise viral peptides that remain conserved across variants for inclusion in universal vaccine candidates. This computational screening has unveiled key immunodominant regions likely to induce cross-protective immunity​, guiding experimental vaccine design and speeding the development of variant-proof immunotherapies. (H. Deng et al., 2025). Many studies in silico were on the spike protein, and a small subset of SARS-CoV-2 proteins (Sankhala et al., 2024). The primary objective of this study is to identify conserved and immunogenic regions across the SARS-CoV-2 genome using an immunoinformatic approach. The secondary aim of this study is to determine the allergenicity of the conserved epitopes to screen out the best possible, reliable candidates for broad-spectrum vaccines and monoclonal antibody therapy targets. Methods Identification of conserved regions in the SARS-CoV-2 genome One lakh thirty-three thousand one hundred and fifty-four (1,33,154) SARS-CoV-2 genome sequences from Indian patients deposited from 1st September 2021 to 31st March 2023 were collected from the GISAID database. The total number of mutations was obtained from analysing data through the R package. R libraries like seqinR (4.2.8) (29), protr (1.6.2) (30) and Biostrings (2.58.0) (31) were used to analyse this data. Regions with a minimum of 8 amino acid length that showed no mutation were considered conserved sequences. The reference genome was obtained from the NCBI accession number: NC_045512.2. The frequency of the mutations in any given position was also calculated. Nearly every single position had a mutation, and no conserved regions could be found. To account for that, we then assumed that mutations with less than 0.1% of the maximum frequency of a protein were rare in the population or sequencing errors. The frequency of mutations for every single position (for each protein) is calculated, and the maximum frequency is identified. 0.1% of this maximum frequency was set as the cut-off, and everything below this was assumed to be zero. The presence or absence of a mutation at any given position was set to binary values, 1 (presence of mutation) and 0 (absence of mutation). Regions with more than eight consecutive zeroes were identified, and their corresponding protein sequence was determined (Fig. 1 ). Immunogenicity and protein modelling Antigenicity and allergenicity of the identified conserved regions were determined as follows. VaxiJen tool was used to identify immunoprotective sequences based on identifying such sequences from bacterial, viral, tumour, fungi and parasite antigens and calculating their auto-cross covariance values (Doytchinova & Flower, 2007). The threshold for viral models was 0.4 (default value); sequences that scored equal to or below 0.4 were considered non-antigenic, and those that scored above were considered antigenic. The Immune Epitope Database (IEDB) tools were used to find possible T-cell and B-cell epitopes in conserved protein sequences. From the input sequence, it identifies the string most likely to have immunogenicity and assigns a score of likelihood (Vita et al., 2025). The NetCTL 1.2 tool was used to identify CTL epitopes in the amino acid sequences of SARS-CoV-2. It supports 12 MHC class 1 supertypes (Larsen et al., 2007). The allergenicity of the amino acid sequences was identified using the AllerTOP tool. Only those sequences with no allergenic effects were used for further analysis (Dimitrov et al., 2014). The conserved antigenic and non-allergenic epitopes were then mapped onto the respective crystal structure of the proteins as follows. Experimentally determined structures of non-structural proteins (NSPs) and structural proteins of SARS-CoV-2 were retrieved from the RSCB Protein Data Bank (PDB) (Berman et al., 2000). The structure of conserved peptide sequences was visualised in PyMOL v2.4.1 (Seeliger & De Groot, 2010). The structures of NSP-4 and NSP-6 were predicted using the Alphafold2 v2.1.1, which uses a machine learning approach for modelling even without homologous structures (Jumper et al., 2021). The best model was selected from the five predicted structures based on the predicted local distance difference test (PLDDT) score. Further, the model was evaluated using the PROCHECK [18] and ERRAT [19] modules of the SAVES v6.0 server to validate the stereochemistry and overall quality factors. The loop regions of the predicted models were refined using the ModLoop server(Fiser & Sali, 2003) and validated using the SAVES server. Modelled structures were analysed using PyMOL. Results and Discussion The analysis revealed 62,94,995 total mutations, including 66,861 unique mutations across the SARS-CoV-2 genome in the Indian strains (Table 1). When data was analysed directly, no conserved regions were detected. Then, an algorithm was employed, assuming that the mutations below a certain “level” would be zero. Several trials were conducted, with cut-off numbers, namely “below 5 (whole number)”, “below 10 (whole number)”, “0.1% of highest frequency”, and “1% of highest frequency” across multiple clades and lineages during the preliminary stage. It was observed that “0.1% of highest frequency” gave a reasonable number of conserved sequences. Therefore, the algorithm used was that all sequences comprising at least eight consecutive amino acids with mutation frequencies above 0.1% were classified as conserved regions in this study (Supplementary Table 1). SARS-CoV-2 viral proteins were then analysed for conserved sequences to identify areas of potential clinical significance. The analysis focused on identifying highly conserved regions across different variants to ensure broad applicability. A total of 270 conserved sequences were identified throughout the SARS-CoV-2 genome (Supplementary Table 1). These sequences were then assessed for their antigenicity and allergenic properties. Among these, 73 antigenic and non-allergenic residues were identified throughout the proteome (Table 2, Supplementary Table 1, Supplementary Table 2). These findings provide insights into the stability and evolutionary conservation, suggesting their probable crucial role in developing robust detection assays with targeted therapeutic and preventive interventions. Table 1 Number of conserved, antigenic and non-allergenic sequences in each of the SARS-CoV-2 proteins. S.No Protein Length of the protein (aa) No. of conserved sequences No. of antigenic and non-allergenic sequences 1 Spike 1273 49 11 2 Envelope 75 2 1 3 Membrane 222 7 4 4 Nucleocapsid 419 14 2 5 NSP-1 180 7 2 6 NSP-2 638 0 0 7 NSP-3 1945 59 21 8 NSP-4 500 20 6 9 NSP-5 306 11 2 10 NSP-6 290 9 4 11 NSP-7 83 0 0 12 NSP-8 198 1 0 13 NSP-9 113 0 0 14 NSP-10 139 0 0 15 NSP-12 932 31 6 16 NSP-13 601 24 6 17 NSP-14 527 22 6 18 NSP-15 346 14 2 19 NSP-16 298 0 0 Table 2 Number of mutations and unique mutations observed across the SARS-CoV-2 genome S No Protein Length (AA) Total No. of mutations No. of Unique mutations AA Position with most mutations and the mutation Frequency of highest mutation 1 Spike 1273 2875012 12929 D614G 131707 2 Envelope 75 108563 481 T9I 87662 3 Membrane 222 237591 1801 A63T 88148 4 Nucleocapsid 419 679718 3811 P13L 86041 5 NSP1 180 94446 1177 S135R 76750 6 NSP2 638 45316 4556 P129L 1581 7 NSP3 1945 458711 18134 G489S 79123 8 NSP4 500 428822 4556 T492I 119745 9 NSP5 306 117695 2131 P132H 89191 10 NSP6 290 262791 2080 G107del 57094 11 NSP7 83 1625 317 M75I 74 12 NSP8 198 20068 926 N118S 12832 13 NSP9 113 3826 380 T35I 375 14 NSP10 139 6023 610 M122V 1209 15 NSP12 932 224130 5698 P323L 129979 16 NSP13 601 168140 3740 R392C 80369 17 NSP14 527 153419 3371 I42V 86910 18 NSP15 346 92205 2562 T112I 73896 19 NSP16 298 11520 2092 K160R 800 The conserved, antigenic, and non-allergenic residues identified in each SARS-CoV-2 protein were subsequently mapped onto corresponding homology models derived from their crystal structures. The functional significance of each residue is detailed below. Spike Protein : Our analysis revealed that the Spike (S) protein, which is 1273 amino acids long, contains 49 conserved regions (Table 2, Supplementary Table 1). Of these, 11 were found to be antigenic and non-allergenic peptides (Table 2, Supplementary Table 1, Fig. 2A). Identifying conserved regions within the S protein is pivotal for developing broad-spectrum antivirals and vaccines. Despite the high mutation rate observed in various S protein domains, certain regions remain highly conserved, underscoring their functional importance. The N-terminal conserved domain (NTD) peptide (293–338) is implicated in host-cell recognition and has been identified as a target for neutralising antibodies (X. Xia, 2021). Mutations in this region can contribute to immune evasion; however, conserved epitopes within the NTD may serve as viable targets for vaccine development (X. Xia, 2021). The S protein’s Receptor Binding Domain (RBD) is critical for binding to the ACE2 receptor, facilitating viral entry into host cells (Tian et al., 2021). While the RBD is subject to frequent mutations, specific conserved sequences within this domain are essential for maintaining its structural integrity and function. One of the identified conserved peptides is mapped (510–519) in the RBD. Notably, the N501Y mutation has strengthened the binding affinity between the spike protein and ACE2 receptor, enhancing viral infectivity (Tian et al., 2021). The H519N mutation significantly decreases SARS-CoV-2 replication in human lung epithelial cells and reduces infectivity in pseudotyped virus (Cereghino et al., 2024). Targeting these conserved regions could lead to the development of broad-spectrum neutralising antibodies (Huan et al., 2024). The fusion peptide of the spike (S) protein plays a critical role in mediating the fusion between the viral and host cell membranes, an essential step in viral entry (Hoffmann et al., 2020). The high conservation of fusion peptide sequences across various coronavirus strains suggests their potential as universal vaccine targets. Peptide-based fusion inhibitors derived from these conserved sequences have demonstrated efficacy against emerging coronaviruses (S. Xia et al., 2014). We have identified conserved regions (693–700, 705–746, 748–763) in this region, suggesting these peptides’ functional significance. S protein’s heptad repeat (HR) regions are integral to the conformational changes required for membrane fusion and viral entry (S. Xia et al., 2019). Mimetic proteins structurally imitating the HR1 region in a trimeric coiled-coil conformation showed potential in inhibiting SARS-CoV-2 infection in vitro (Zhu et al., 2020). Our analysis revealed three conserved peptides (1021–1039, 1041–1069, 1092–1100) in this region. Targeting these conserved regions within the S protein is a strategic approach to developing effective therapeutics and vaccines across multiple coronavirus strains. Focusing on these regions makes it possible to design interventions that maintain efficacy even as the virus undergoes mutations in other regions. Envelope Protein : The SARS-CoV-2 envelope (E) protein, comprising 75 amino acids, plays a crucial role in virus assembly, budding, and pathogenesis. The conserved antigenic and non-allergenic sequence (residues 36–48) is located within the hydrophobic transmembrane domain (TMD) of the E protein, which is essential for anchoring it to the viral envelope (Fig. 2B, Table 2) (Schoeman & Fielding, 2019). Structural studies have shown that the E protein's TMD forms a pentameric helical bundle, creating a cation-selective ion channel critical for viral pathogenicity, highlighting its role as a potential target for antiviral drugs (Hong et al., 2020; Schoeman & Fielding, 2019). This underscores the TMD's potential as a therapeutic intervention target for hindering virus assembly and release. Targeting this region could lead to the design of inhibitors that disrupt E protein function, thereby mitigating SARS-CoV-2 infectivity and pathogenicity. Membrane Protein: The SARS-CoV-2 membrane (M) protein, comprising 222 amino acids, plays a vital role in viral assembly, morphogenesis, and pathogenesis (Z. Zhang et al., 2022). It is the most abundant structural component of the virus and interacts with other essential proteins, such as S and E proteins, to regulate viral budding and genome encapsulation (Z. Zhang et al., 2022). Four of the seven conserved regions observed in the current study were found to be antigenic and non-allergenic (Fig. 2C, Table 2). The transmembrane location of the conserved region (residues 20–27) could contribute to its anchoring within the viral envelope and is essential for membrane curvature during viral budding (Neuman et al., 2011). Hydrophobic interactions within this region facilitate lipid bilayer integration, ensuring the stability of the viral structure (Dolan et al., 2022). Another conserved region (residues 40–62) is known to be critical for protein-protein interactions involved in viral assembly (Z. Zhang et al., 2022). Studies suggest that mutations in this domain disrupt virion formation, producing non-infectious viral particles (Siu et al., 2008). The conserved region (residues 105–184) is known to interact with the nucleocapsid (N) protein, which plays a key role in encapsulating the viral RNA genome (Siu et al., 2008). Disrupting this region has been shown to impair viral replication and assembly, making it a key target for antiviral drug development (Siu et al., 2008). The host-cell interaction domain region (residues 186–206) plays a fundamental role in binding to host cell membranes and is involved in the budding process of new virions (Siu et al., 2008). Computational docking studies suggest that peptides mimicking this domain could act as viral assembly inhibitors, preventing new virions from exiting the host cell (Neuman et al., 2011). Given its role in viral structure and function, the M protein is a key therapeutic intervention target. Conserved sequences within the M protein offer promising opportunities for developing antiviral agents that can disrupt viral assembly and budding (Neuman et al., 2011). In addition, targeting the M protein interactions with S and E proteins could prevent the formation of fully functional virions, reducing viral spread within infected individuals (Boson et al., 2021). Moreover, the immunogenic properties of the M protein suggest its potential use in vaccine formulations. The conserved nature of the M protein ensures broad-spectrum vaccine effectiveness, even against emerging SARS-CoV-2 variants. Nucleocapsid Protein: The nucleocapsid (N) protein of SARS-CoV-2, a crucial structural component, is involved in RNA binding, genome packaging, and viral replication (Kang et al., 2020). Comprising 419 amino acids, the N protein exhibited 14 conserved regions, with two areas being antigenic and non-allergenic, underscoring its evolutionary stability across different variants. This conservation suggests a crucial functional significance, making the N protein a prime target for diagnostic and therapeutic applications. Both the conserved antigenic residues (residues 102–118 and residues 301–318) (Fig. 2D, Table 2) are particularly noteworthy due to their potential involvement in RNA binding and protein-protein interactions (Cubuk et al., 2021). The first conserved peptide sequence (residues 102–118) falls within a highly structured region, which may contribute to the protein's ability to interact with viral and host components (Cubuk et al., 2021). Similarly, the second conserved peptide sequence (residues 301–318) is positioned within a flexible domain that may facilitate dynamic conformational changes essential for nucleocapsid function (Cubuk et al., 2021; Dinesh et al., 2020). The high conservation of these regions across SARS-CoV-2 variants suggests their indispensable role in viral assembly and propagation. The N protein is highly immunogenic so that these conserved sequences may serve as potential epitopes for monoclonal antibody development and vaccine design (Dutta et al., 2020). Additionally, the involvement of the identified conserved sequences in viral RNA replication processes suggests that targeting them with small molecules or peptide inhibitors could disrupt viral replication, offering a promising therapeutic avenue (Iserman et al., 2020). The N protein remains a primary target for rapid antigen detection tests in diagnostic applications due to its abundance during infection. Monoclonal antibodies directed against the identified conserved regions could enhance the sensitivity and specificity of lateral flow assays, aiding in early and reliable SARS-CoV-2 detection (Grant et al., 2021). In conclusion, the remarkable conservation of these two regions within the SARS-CoV-2 N protein highlights their functional importance in viral pathogenesis. Future studies should focus on elucidating their structural and functional roles in greater detail, paving the way for improved antiviral strategies and diagnostic tools. Non-structural protein 1: NSP-1 is a key virulence factor in suppressing host immune responses and facilitating viral replication (Thoms et al., 2020). Of the seven conserved regions in the NSP-1, two peptides were found to be antigenic and non-allergenic. One of the peptides (residues 50–59) was mapped onto the surface of the NSP-1 (Fig. 3A, Table 2). This region (residues 50–59) within the N-terminal domain (NTD) of SARS-CoV-2 NSP-1 may play a role in host immune suppression and viral replication. Structurally, this region forms part of the β2–β3 loop, contributing to the electrostatic surface properties of the NTD and stabilising its interactions with host factors (Li et al., 2023). The acidic patch formed by residues E55 and E57 is located adjacent to the α1-helix and may play a role in the selective binding of viral RNA, while simultaneously inhibiting host mRNA translation (Thoms et al., 2020). Functionally, NSP-1 inhibits host gene expression by blocking translation and suppressing mRNA transport (Schubert et al., 2020). Mutational studies indicated that alterations in the 50–59 loop impair NSP-1’s ability to suppress host defences, leading to attenuation of viral replication (Lapointe et al., 2021). Deletion or mutation of residues in this region has been linked to reduced cytotoxicity and loss of host shutoff function, suggesting its importance in NSP-1’s virulence mechanism (Kamitani et al., 2009). Additionally, attenuated viral strains with targeted mutations in this region may serve as candidates for live-attenuated vaccine development (Lapointe et al., 2021). This conserved region presents a promising therapeutic target for antiviral strategies and vaccine design by enabling immune evasion and viral replication. Non-structural protein 3: The NSP-3 of SARS-CoV-2 is reported to play a role in viral replication and the modulation of host immune responses (Lei et al., 2018). In our study, NSP-3 is the protein identified with the maximum number of conserved residues with antigenic significance (N = 59, N = 21, respectively) (Table 2, Fig. 3B). Other researchers analysed several key amino acid sequences within NSP-3 for their structural and functional significance in viral pathogenesis (Low et al., 2022). The N-terminal region, consisting of residues 1–8, plays a crucial role in the early stages of viral replication and is also associated with ubiquitin-like domain 1. This domain has been implicated in protein-protein interactions that may modulate host immune responses (Gao et al., 2020). Similarly, residues 96–107 are located within the SARS-Unique Domain (SUD), a region unique to SARS-related coronaviruses, which interacts with host cell proteins and may influence viral replication efficiency (Lei et al., 2018). The papain-like protease (PLPro) domain, containing residues 356–363, plays a pivotal role in cleaving the viral polyprotein and has de-ubiquitinating functions that help SARS-CoV-2 evade immune detection (Bailey-Elkin et al., 2017). Structural studies have highlighted the significance of PLPro in counteracting host antiviral responses, making it a key therapeutic target (Shin et al., 2020). Additionally, the macrodomain 1 of NSP-3, which includes residues 435–461, has been identified as essential in interfering with the host's ADP-ribosylation signalling. This domain is vital for viral replication and immune evasion, with structural analyses emphasising its potential as a drug target (Frick et al., 2020). The transmembrane regions of NSP-3, represented by residues 535–557 and 846–859, contribute to double-membrane vesicles (DMVs), which provide a protected niche for viral RNA synthesis (Wolff et al., 2020). These domains interact with NSP-4 and NSP-6, highlighting their role in viral replication complex formation. The SUD contains multiple functionally relevant sequences, residues 740–748 and 750–770, which are implicated in host-pathogen interactions and may influence the efficiency of viral replication (Tan et al., 2021). Studies suggest that SUD enhances viral pathogenicity through interactions with cellular proteins, further supporting its significance in SARS-CoV-2 infection dynamics. Further downstream, residues 1844–1866 and 1893–1945 are located in regions that may contribute to NSP-3's overall structural stability and interactions with other viral and host components(Lei et al., 2018). Their functional implications warrant further investigation, particularly in viral replication and immune evasion mechanisms. The comprehensive analysis of these NSP-3 sequences provides valuable insights into their functional roles in SARS-CoV-2 pathogenesis. Given their involvement in viral replication, immune modulation, and host interactions, these sequences are potential targets for antiviral drug development. Further structural and biochemical studies must elucidate their full mechanistic contributions and therapeutic potential. Non-structural protein 4: SARS-CoV-2 non-structural protein 4 (NSP-4) is crucial for membrane remodelling during viral replication, interacting with NSP-3 to promote the formation of double-membrane vesicles (DMVs) (Angelini et al., 2013; Oudshoorn et al., 2017). Our analysis revealed 20 conserved residues, with 6 having antigenic potential (Table 2, Fig. 3C). These six conserved motifs in NSP-4, including residues 61–94, 97–111, 129–136, 138–145, 253–263, 387–400, have been shown to possess functional importance (C. J. Gordon et al., 2020). Structurally, these conserved sequences map to key domains, including luminal loops involved in membrane curvature (61–145, 253–263), transmembrane helices forming the DMV-spanning pore (387–400), and the cytosolic C-terminal tail critical for replication complex assembly (493–500) (Neuman, 2016; Zimmermann et al., 2023). Notably, N-glycosylation at Asn131 and multiple disulfide bonds within luminal loops suggest an additional layer of regulation influencing NSP-4 stability and interaction with NSP-3 (Oostra et al., 2008). Despite NSP-4’s high conservation, the T492I mutation, which emerged in Delta and Omicron variants, enhanced viral replication by increasing 3CLpro processing efficiency (X. Lin et al., 2023). This highlights how even minor mutations in NSP-4 can impact viral fitness. Given its critical role in DMV formation, NSP-4 represents an attractive antiviral target, particularly disrupting the NSP–3–NSP–4 interface or DMV-spanning pore (V’kovski et al., 2021). Future studies should explore small molecules or antibodies targeting NSP4, given its low mutation rate compared to other viral proteins, making it a viable broad-spectrum target across coronaviruses. Non-structural protein 5: The NSP-5 main protease (Mpro) of SARS-CoV-2 plays a pivotal role in viral replication by cleaving polyproteins (pp1a/pp1ab) into functional non-structural proteins (L. Zhang et al., 2020). The enzyme is highly conserved, with key sequence segments 36–74 and 248–259 demonstrating strong purifying selection across SARS-CoV-2 variants and related coronaviruses (C. J. Gordon et al., 2020). Interestingly, in our study of the 11 conserved residues identified, these two have antigenic potential (Table 2, Fig. 3D). The 36–74 region contributes to the catalytic domain (domain I) and harbours His41, a critical residue forming the His41–Cys145 catalytic dyad essential for substrate cleavage and viral replication (Zhou et al., 2020). This segment also defines the substrate-binding pocket and interacts with known inhibitors such as Paxlovid (nirmatrelvir) (Owen et al., 2021). The 248–259 region within domain III is crucial for dimerisation, an essential step in NSP-5 activation (Kneller et al., 2020). Disrupting this interface destabilises the enzyme, leading to a loss of function and viral replication inhibition (Su et al., 2022). Despite the emergence of SARS-CoV-2 variants of concern, NSP-5 remains highly conserved, with minimal mutations in its active site or dimerisation interface (Sacco et al., 2020). Mutational studies confirm that substitutions in these regions impair viral fitness, highlighting their functional importance (Jin et al., 2020). Given the stability of these sequences, Mpro remains a prime antiviral target. Covalent inhibitors like nirmatrelvir and peptidomimetics have been designed to irreversibly bind the active site, effectively blocking viral replication (Owen et al., 2021). Additionally, allosteric inhibitors targeting the dimerisation interface have shown promise, offering an alternative antiviral approach (Kubra et al., 2023). Given these segments’ extreme conservation and functional indispensability, targeting NSP5 with direct-acting antivirals provides a robust therapeutic strategy against SARS-CoV-2 and future coronavirus outbreaks (L. Zhang et al., 2020). Future research should focus on dimer interface inhibitors to complement existing active-site-targeting drugs, ensuring broad-spectrum efficacy against coronaviruses. Non-structural protein 6: The NSP-6 of SARS-CoV-2 promotes the virus's ability to remodel host cell membranes, facilitating DMVs essential for viral RNA replication. This function is conserved across coronaviruses, underscoring the importance of specific sequences within NSP-6 (Benvenuto et al., 2020). We identified nine conserved residues in this protein, with 4 exhibiting antigenic potential (Table 2, Fig. 3E). The sequences with residues 12–23 and residues 25–34 are located within the transmembrane domains of NSP-6, which are highly conserved among SARS-CoV-2 variants and related coronaviruses, including SARS-CoV and MERS-CoV. This conservation suggests a fundamental role in membrane association and DMV formation (D. E. Gordon et al., 2020a). Similarly, the sequences with residues 169–179 and residues 222–231 are preserved across various coronavirus species, indicating their critical role in viral replication (Cottam et al., 2014). The transmembrane regions encompassing residues 12–34 are integral to NSP-6’s ability to anchor to the endoplasmic reticulum membrane. This anchorage is vital for inducing autophagosome formation and subsequent DMV development in viral replication (D. E. Gordon et al., 2020b). The structural conservation of these sequences ensures the integrity required for proper membrane curvature and vesicle formation. Residues 169–179 and 222–231 are implicated in protein-protein interactions within the viral replication complex, facilitating the coordination necessary for efficient RNA synthesis (Cottam et al., 2014). Mutations within these conserved regions can significantly impact NSP-6 function. For instance, alterations in the transmembrane domains may disrupt membrane association, hindering DMV formation and attenuating viral replication (Yang et al., 2020). Targeting these conserved sequences with antiviral agents could impair NSP-6’s function, offering a potential therapeutic strategy (Kang et al., 2020). Compounds that disrupt NSP-6’s membrane interactions or their role in autophagosome formation could effectively inhibit viral replication (Benvenuto et al., 2020). The conserved sequences within NSP-6 are integral to modifying host cell membranes for viral replication. Their preservation across coronavirus species highlights their essential role and presents opportunities for targeted antiviral interventions. Given these sequences’ high conservation and functional importance, NSP-6 remains a viable antiviral target, particularly for strategies that disrupt its interactions with host cell membranes and interfere with DMV formation (Kang et al., 2020). Non-structural protein 12: The SARS-CoV-2 NSP-12 functions as the RNA-dependent RNA polymerase (RdRp), a crucial enzyme for viral genome replication (Hillen et al., 2020). We identified 31 conserved residues in this protein, with 6 exhibiting antigenic potential (Table 2, Fig. 4A). These six conserved sequence regions (residues 186–226, 294–322, 401–462, 464–486, 672–693, and 720–738) have been shown to play essential roles in RdRp function, structural stability, and antiviral drug interactions. Residues 186–226 of NSP-12 facilitate nucleotidyl transfer reactions necessary for RNA synthesis and viral RNA capping (Slanina et al., 2021). Key residues within this region participate in ATP and GTP binding, making it an attractive antiviral target(Subissi et al., 2014) Region 294–322 contains a highly conserved Cys/His-rich zinc-binding motif stabilising NSP-12 by coordinating Zn²⁺ ions (Gao et al., 2020). Structural studies indicate that disrupting these zinc-coordinating residues impairs polymerase activity (Q. Wang et al., 2020). Polymerase core regions with residues 401–486, 672–693, 720–738 form key subdomains essential for RNA synthesis. The fingers domain 401–486 contributes to RNA binding and cofactor interactions, particularly with NSP-8, which enhances processivity (Hillen et al., 2020). The palm domain 672–693 contains motif B, a flexible loop that adjusts during nucleotide incorporation, facilitating efficient RNA synthesis (Shannon et al., 2020). The 720–738 region lies adjacent to the active site and stabilises the polymerase’s catalytic domain (Yin et al., 2020). The high conservation of these sequences makes them ideal targets for antiviral drugs. Remdesivir, a nucleotide analogue, binds to the polymerase active site and disrupts RNA synthesis by causing delayed chain termination (C. J. Gordon et al., 2020). Structural studies have shown that remdesivir interacts with conserved residues in the fingers and palm domains, stabilising an inactive polymerase complex (Kokic et al., 2021). Molnupiravir and favipiravir also exploit conserved residues to induce lethal mutagenesis (Agostini et al., 2018). These regions remain highly conserved across SARS-CoV, MERS-CoV, and other coronaviruses, indicating strong evolutionary constraints (Pachetti et al., 2020). However, mutations such as P323L (interface domain) and G671S (motif B loop) have emerged in SARS-CoV-2 variants, potentially enhancing viral replication efficiency (S. M. Kim et al., 2023). Nevertheless, remdesivir resistance mutations (e.g., F480L, V557L) remain rare due to the functional constraints on these conserved regions(Agostini et al., 2018) Overall, the conserved sequences in NSP-12 are essential for viral replication and represent key targets for developing antiviral drugs. Their limited mutational tolerance reinforces their potential as drug-binding sites, highlighting the importance of continued structural and functional studies to optimise therapeutic strategies. Non-structural protein 13: The SARS-CoV-2 NSP-13 is a highly conserved helicase that plays a critical role in viral replication by unwinding RNA and hydrolysing ATP (Jia et al., 2019). We identified 24 conserved residues in this protein, with 6 exhibiting antigenic potential (Table 2, Fig. 4B). These conserved sequences in NSP-13, including residues 1–17, 54–76, 141–153, 414–430, 432–443, and 554–575, correspond to essential functional motifs required for enzymatic activity, structural integrity, and interaction with other viral components. The zinc-binding domain (ZBD), comprising the 1–17 and 54–76 sequences, coordinates Zn²⁺ or Fe–S clusters necessary for NSP-13 stability and RNA unwinding. Studies have shown that mutations in these cysteine-rich motifs disrupt metal coordination and abolish enzymatic function (Maio et al., 2024). The stalk domain (141–153) supports the helicase core and facilitates interactions with NSP-12 and NSP-8 in the replication-transcription complex (J. Chen et al., 2020). The helicase core, including 414–430 (RecA1 domain), 432–443 (RecA2 domain), and 554–575 (motif VI), contains essential ATP-binding and RNA-binding motifs. Motif VI, particularly Arg567 within the 554–575 segment, functions as an "arginine finger," crucial for ATP hydrolysis and energy transduction (Hao et al., 2017). Structural studies have revealed that these conserved sequences undergo conformational changes upon ATP binding, facilitating RNA unwinding (Jia et al., 2019). NSP13 is a promising antiviral target due to its high conservation across coronaviruses. Small-molecule inhibitors such as bismuth complexes, flavonoids (myricetin, scutellarein), and SSYA10-001 have been shown to target these conserved motifs, disrupting ATPase and helicase activity (Adedeji et al., 2012). Additionally, the ZBD is a druggable site, as bismuth-based compounds destabilise its zinc-finger motifs, effectively inhibiting the helicase (Shu et al., 2020). Comparative sequence analysis across SARS-CoV, MERS-CoV, and other coronaviruses highlights the strong evolutionary conservation of these motifs, underscoring their indispensable role in viral replication, making them attractive targets for therapeutics and vaccines. Non-structural protein 14: The NSP-14 of SARS-CoV-2 is integral to the virus's replication fidelity and immune evasion strategies (Y. Chen et al., 2009; Ogando et al., 2020). Our sequence analysis has identified 22 conserved regions within NSP-14, including six antigenic residues: 32–41, 50–66, 126–139, 145–156, 223–249, and 361–370 (Table 2, Fig. 4C). The conservation of these sequences across various SARS-CoV-2 isolates suggests their critical role in maintaining the structural integrity and enzymatic functions of NSP-14 (Saikatendu et al., 2005). Notably, NSP-14 of SARS-CoV and SARS-CoV-2 share over 95% amino acid sequence similarity, underscoring their evolutionary conservation and potential as a therapeutic target (Robson et al., 2020). The ExoN activity of NSP-14 is essential for correcting errors during RNA synthesis, thereby reducing the mutation rate and contributing to the stability of the viral genome (Bouvet et al., 2010). Additionally, the N7-MTase domain's role in mRNA capping protects viral RNA from host immune responses, facilitates efficient translation, and ensures viral proliferation (S. Lin et al., 2020). The crystal structure of SARS-CoV-2 NSP-14 demonstrates that the ExoN domain's enzymatic activity is metal ion-dependent, preferably utilising Mg²⁺, and that the N7-MTase domain harbours a conserved DxG motif for S-adenosyl-L-methionine binding, characteristic of coronaviruses (Saikatendu et al., 2005). Non-structural protein 15: The SARS-CoV-2 NSP-15 is an endoribonuclease highly conserved among coronaviruses and plays a crucial role in viral RNA processing and immune evasion (Y. Kim et al., 2020). It cleaves viral RNA at uridine sites, preventing the accumulation of immunostimulatory dsRNA, thereby helping the virus evade host immune responses (X. Deng et al., 2017). We identified 14 conserved residues in this protein, with 2 exhibiting antigenic potential (Table 2, Fig. 4D). The conserved residues 1–9 and 321–336 are functionally significant as they contribute to NSP-15's hexameric structure and catalytic activity, making them potential antiviral drug targets ((Saramago et al., 2022). The peptide with residues 1–9 at the N-terminus is part of the oligomerisation domain, essential for forming the active hexameric complex (Y. Kim et al., 2020). Mutations in this region impair hexamerization and lead to loss of enzymatic activity, suggesting its importance in structural integrity (Ivanov et al., 2004) The conservation of this motif across coronaviruses highlights its critical role in viral replication and potential as a drug target (X. Deng et al., 2017). The peptide with residues 321–336 in the C-terminal region is located near the active site of NSP-15 and is highly conserved across beta-coronaviruses (Saramago et al., 2022). It contributes to substrate recognition and enzymatic function, ensuring efficient RNA cleavage (Saramago et al., 2022). Structural studies show that this sequence forms part of the uridine-binding pocket, crucial for its RNA endonuclease activity (Hackbart et al., 2020). Mutations in this region disrupt catalytic efficiency, accumulating dsRNA and increasing host immune activation (X. Deng et al., 2017). Viruses lacking a functional NSP-15 enzyme show reduced replication efficiency and are more susceptible to host immune responses. The conserved residues 1–9 and 321–336 in SARS-CoV-2 NSP-15 are structurally and functionally crucial. They contribute to enzyme oligomerisation, substrate recognition, and RNA processing, ensuring viral replication and immune evasion. Their high conservation makes them ideal targets for antiviral drug development. Conclusions In conclusion, our findings provide insights into the stability and evolutionary conservation of SARS-CoV-2 protein sequences, suggesting their probable crucial role in developing robust detection assays and targeted therapeutic and preventive interventions. These conserved sequences within SARS-CoV-2 are integral for viral replication and immune evasion. Mutations within these sequences are rare due to their functional constraints, and the reports of experimental mutagenesis studies confirm that disruptions in these regions would lead to loss of viral replication efficiency, making them attractive therapeutic and preventive targets for broad-spectrum SARS-CoV-2 variants. Future research may focus on developing inhibitors that exploit these conserved sites to disrupt viral replication, effectively providing effective therapeutic and prophylactic strategies against SARS-CoV-2 and related coronaviruses. Abbreviations 1. SARS-CoV-2: Severe acute respiratory syndrome-coronavirus-2 2. COVID-199: Corona virus disease-19 3. S protein: Spike protein 4. RBD: Ribosome binding domain 5. HR: Heptad repeat 6. E protein: Envelope protein 7. TMD: Trans membrane domain 8. M protein: Membrane protein 9: NSP: Non-structural protein Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and material: The datasets used/analysed during the current study are available in the NISAID repository, https://gisaid.org Competing interests: The authors declare that they have no competing interests Funding: No funding was received for this study Authors’ contributions: RM, KP, JS, and SR contributed to the study conception and design. JS, SR, ST, BRN, ARA, SKP, AV, JP, VK, MMA, MM, MY, and PKM performed material preparation and data collection. RM, KP, PSD, and AK analysed the data. The first draft of the manuscript was written by RM, KP, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pavan","middleName":"Kumar","lastName":"Muttineni","suffix":""},{"id":471497399,"identity":"43b70041-9cba-43a2-a6e3-4f8bf1bf00f3","order_by":15,"name":"Pankaj Singh Dolaniya","email":"","orcid":"","institution":"University of Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"Singh","lastName":"Dolaniya","suffix":""},{"id":471497400,"identity":"527759ba-5931-491e-877f-2865a515964f","order_by":16,"name":"Anand Kumar Kondapi","email":"","orcid":"","institution":"University of Hyderabad","correspondingAuthor":false,"prefix":"","firstName":"Anand","middleName":"Kumar","lastName":"Kondapi","suffix":""}],"badges":[],"createdAt":"2025-06-02 05:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6798845/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6798845/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84781313,"identity":"095cf97c-5505-4006-89a0-1f60744e5a6c","added_by":"auto","created_at":"2025-06-17 09:35:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131094,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of the analysis workflow\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/ae61fcd29b994cc41ce0ce79.png"},{"id":84781316,"identity":"3e7367f5-9aa4-4330-9dba-145cf366342a","added_by":"auto","created_at":"2025-06-17 09:35:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1249689,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of immunogenic conserved peptide residues onto SARS-CoV-2 structural protein homology models.\u003c/p\u003e\n\u003cp\u003eAntigenic and non-allergenic residues identified were mapped onto Spike (A), Envelope (B), Membrane (C), and Nucleocapsid (D) homology models. Residues mapped on the surface of the homology models are depicted.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/c911f6bf9453c3449a13c00c.png"},{"id":84782294,"identity":"a42d64ea-99d9-46df-b191-92c6beda39c2","added_by":"auto","created_at":"2025-06-17 09:43:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1252507,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of immunogenic conserved peptide residues onto SARS-CoV-2 non-structural protein (located on the Orf1-a of the SARS-CoV-2 genome) homology models.\u003c/p\u003e\n\u003cp\u003eAntigenic and non-allergenic residues identified were mapped onto NSP-1 (A), NSP-3 (B), NSP-4 (C), NSP-5 (D), and NSP-6 (E) homology models. Residues mapped on the surface of the homology models are depicted.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/74ccd977a9bfd7579bf687ec.png"},{"id":84782752,"identity":"b42db2ca-80d1-4ace-90f3-099945975730","added_by":"auto","created_at":"2025-06-17 09:51:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1103436,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of immunogenic conserved peptide residues onto SARS-CoV-2 non-structural protein (located on the Orf1-b of the SARS-CoV-2 genome) homology models.\u003c/p\u003e\n\u003cp\u003eAntigenic and non-allergenic residues identified were mapped onto NSP-12 (A), NSP-13 (B), NSP-14 (C), and NSP-15 (D) homology models. Residues mapped on the surface of the homology models are depicted.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/0f1cc2a7ef290ae615f1d7cd.png"},{"id":85500397,"identity":"29e35eb9-a3c8-40b7-9541-223d01e71e52","added_by":"auto","created_at":"2025-06-26 14:32:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5281448,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/2b0c96ef-567c-4d57-9399-dff795fed664.pdf"},{"id":84781314,"identity":"abd79449-9d58-4f1d-a01b-33dfe8e9d078","added_by":"auto","created_at":"2025-06-17 09:35:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":97821,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1: Details of the conserved, antigenic, and non-allergenic residues identified in the different proteins of the SARS-CoV-2 genome.\u003c/p\u003e","description":"","filename":"Suppementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/3bebe32bd494cc3d9c404071.xlsx"},{"id":84782293,"identity":"959915bd-3685-4fd0-a27c-17d5d70b1dce","added_by":"auto","created_at":"2025-06-17 09:43:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27913,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2: Summary of the sequences of the conserved antigenic and non-allergenic residues identified in the different proteins of the SARS-CoV-2 genome.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6798845/v1/5f585c6c569d3f1ee7c9f726.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Conserved SARS-CoV-2 Viral Peptides as Potential Prophylactic and Therapeutic Targets","fulltext":[{"header":"Background","content":"\u003cp\u003e​ The rapid emergence of SARS-CoV-2 variants has challenged the efficacy of first-generation vaccines. For instance, the heavily mutated Omicron variant evades many neutralising antibodies from prior infection or vaccination (Choi et al., 2022). This immune escape has led to breakthrough infections and prompted the rollout of variant-updated booster vaccines (e.g. bivalent BA.4/5 and monovalent XBB.1.5 boosters) to restore protection (Muik et al., 2024). Emerging COVID-19 vaccines increasingly focus on conserved viral epitopes to achieve broader protection. Strategies include multi-epitope vaccines incorporating conserved B- and T-cell epitopes across the SARS-CoV-2 proteome(C. Y. Wang et al., 2023a) The SARS-CoV-2 genome is approximately 30,000 nucleotides long and encodes 29 proteins, with four structural proteins, 16 non-structural proteins (NSPs), and nine accessory proteins (Bai et al., 2021). Recent studies revealed that including peptides from internal proteins like nucleocapsid (N) and membrane (M) alongside Spike (S) elicit robust CD4⁺ and CD8⁺ T-cell responses to complement neutralising antibodies. These peptides are also reported to induce potent and long-lasting B- and T-cell immunity in clinical trials, suggesting that they confer broad protection against diverse variants (Hsieh et al., 2024; Prakash et al., 2024a; Sankhala et al., 2024; C. Y. Wang et al., 2023b). T-cell-mediated immunity has proven more resilient to viral variation; studies show that most T-cell epitopes remain unchanged across variants (Prakash et al., 2024b). Notably, \u0026gt;\u0026thinsp;95% of CD8⁺ T-cell epitopes identified in the original strain are still conserved in Omicron (Choi et al., 2022). This preservation of conserved peptide targets helps maintain T-cell recognition and protection against severe disease despite the antigenic drift of SARS-CoV-2 (Choi et al., 2022). These reports underscore why next-generation vaccines are pivoting to include conserved elements to confer immunity that endures against current and future variants​ (Prakash et al., 2024b). Computational immunology has been instrumental in pinpointing conserved SARS-CoV-2 peptide targets. Immuno-informatics leverages the growing genomic data from SARS-CoV-2 variants and related coronaviruses to map regions of high sequence conservation. These conserved epitopes are often functionally important; mutation-intolerant regions are attractive targets for the broad-spectrum vaccine and serve as potential regions for the design of diagnostic and therapeutic approaches​. In silico tools also predict the binding of these epitopes to the common human MHC alleles, ensuring broad population coverage (Prakash et al., 2024b)​. By integrating sequence alignment, structural modelling, and epitope prediction algorithms, researchers can efficiently prioritise viral peptides that remain conserved across variants for inclusion in universal vaccine candidates. This computational screening has unveiled key immunodominant regions likely to induce cross-protective immunity​, guiding experimental vaccine design and speeding the development of variant-proof immunotherapies. (H. Deng et al., 2025).\u003c/p\u003e \u003cp\u003eMany studies in silico were on the spike protein, and a small subset of SARS-CoV-2 proteins (Sankhala et al., 2024). The primary objective of this study is to identify conserved and immunogenic regions across the SARS-CoV-2 genome using an immunoinformatic approach. The secondary aim of this study is to determine the allergenicity of the conserved epitopes to screen out the best possible, reliable candidates for broad-spectrum vaccines and monoclonal antibody therapy targets.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of conserved regions in the SARS-CoV-2 genome\u003c/h2\u003e \u003cp\u003eOne lakh thirty-three thousand one hundred and fifty-four (1,33,154) SARS-CoV-2 genome sequences from Indian patients deposited from 1st September 2021 to 31st March 2023 were collected from the GISAID database. The total number of mutations was obtained from analysing data through the R package. R libraries like seqinR (4.2.8) (29), protr (1.6.2) (30) and Biostrings (2.58.0) (31) were used to analyse this data. Regions with a minimum of 8 amino acid length that showed no mutation were considered conserved sequences. The reference genome was obtained from the NCBI accession number: NC_045512.2. The frequency of the mutations in any given position was also calculated. Nearly every single position had a mutation, and no conserved regions could be found. To account for that, we then assumed that mutations with less than 0.1% of the maximum frequency of a protein were rare in the population or sequencing errors. The frequency of mutations for every single position (for each protein) is calculated, and the maximum frequency is identified. 0.1% of this maximum frequency was set as the cut-off, and everything below this was assumed to be zero. The presence or absence of a mutation at any given position was set to binary values, 1 (presence of mutation) and 0 (absence of mutation). Regions with more than eight consecutive zeroes were identified, and their corresponding protein sequence was determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunogenicity and protein modelling\u003c/h3\u003e\n\u003cp\u003eAntigenicity and allergenicity of the identified conserved regions were determined as follows. VaxiJen tool was used to identify immunoprotective sequences based on identifying such sequences from bacterial, viral, tumour, fungi and parasite antigens and calculating their auto-cross covariance values (Doytchinova \u0026amp; Flower, 2007). The threshold for viral models was 0.4 (default value); sequences that scored equal to or below 0.4 were considered non-antigenic, and those that scored above were considered antigenic. The Immune Epitope Database (IEDB) tools were used to find possible T-cell and B-cell epitopes in conserved protein sequences. From the input sequence, it identifies the string most likely to have immunogenicity and assigns a score of likelihood (Vita et al., 2025). The NetCTL 1.2 tool was used to identify CTL epitopes in the amino acid sequences of SARS-CoV-2. It supports 12 MHC class 1 supertypes (Larsen et al., 2007). The allergenicity of the amino acid sequences was identified using the AllerTOP tool. Only those sequences with no allergenic effects were used for further analysis (Dimitrov et al., 2014). The conserved antigenic and non-allergenic epitopes were then mapped onto the respective crystal structure of the proteins as follows. Experimentally determined structures of non-structural proteins (NSPs) and structural proteins of SARS-CoV-2 were retrieved from the RSCB Protein Data Bank (PDB) (Berman et al., 2000). The structure of conserved peptide sequences was visualised in PyMOL v2.4.1 (Seeliger \u0026amp; De Groot, 2010). The structures of NSP-4 and NSP-6 were predicted using the Alphafold2 v2.1.1, which uses a machine learning approach for modelling even without homologous structures (Jumper et al., 2021). The best model was selected from the five predicted structures based on the predicted local distance difference test (PLDDT) score. Further, the model was evaluated using the PROCHECK [18] and ERRAT [19] modules of the SAVES v6.0 server to validate the stereochemistry and overall quality factors. The loop regions of the predicted models were refined using the ModLoop server(Fiser \u0026amp; Sali, 2003) and validated using the SAVES server. Modelled structures were analysed using PyMOL.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe analysis revealed 62,94,995 total mutations, including 66,861 unique mutations across the SARS-CoV-2 genome in the Indian strains (Table\u0026nbsp;1). When data was analysed directly, no conserved regions were detected. Then, an algorithm was employed, assuming that the mutations below a certain \u0026ldquo;level\u0026rdquo; would be zero. Several trials were conducted, with cut-off numbers, namely \u0026ldquo;below 5 (whole number)\u0026rdquo;, \u0026ldquo;below 10 (whole number)\u0026rdquo;, \u0026ldquo;0.1% of highest frequency\u0026rdquo;, and \u0026ldquo;1% of highest frequency\u0026rdquo; across multiple clades and lineages during the preliminary stage. It was observed that \u0026ldquo;0.1% of highest frequency\u0026rdquo; gave a reasonable number of conserved sequences. Therefore, the algorithm used was that all sequences comprising at least eight consecutive amino acids with mutation frequencies above 0.1% were classified as conserved regions in this study (Supplementary Table\u0026nbsp;1). SARS-CoV-2 viral proteins were then analysed for conserved sequences to identify areas of potential clinical significance. The analysis focused on identifying highly conserved regions across different variants to ensure broad applicability. A total of 270 conserved sequences were identified throughout the SARS-CoV-2 genome (Supplementary Table\u0026nbsp;1). These sequences were then assessed for their antigenicity and allergenic properties. Among these, 73 antigenic and non-allergenic residues were identified throughout the proteome (Table\u0026nbsp;2, Supplementary Table\u0026nbsp;1, Supplementary Table\u0026nbsp;2). These findings provide insights into the stability and evolutionary conservation, suggesting their probable crucial role in developing robust detection assays with targeted therapeutic and preventive interventions.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eNumber of conserved, antigenic and non-allergenic sequences in each of the SARS-CoV-2 proteins.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS.No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLength of the protein (aa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of conserved sequences\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of antigenic and non-allergenic sequences\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpike\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnvelope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMembrane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleocapsid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP-16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eNumber of mutations and unique mutations observed across the SARS-CoV-2 genome\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS No\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLength (AA)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal No. of mutations\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of Unique mutations\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAA Position with most mutations and the mutation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFrequency of highest mutation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpike\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2875012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD614G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e131707\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnvelope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e108563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT9I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87662\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMembrane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e237591\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA63T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNucleocapsid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e679718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3811\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP13L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86041\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS135R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76750\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP129L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1581\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e458711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG489S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79123\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e428822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT492I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e119745\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e306\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e117695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP132H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89191\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e262791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG107del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57094\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM75I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e926\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN118S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12832\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3826\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT35I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e375\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM122V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1209\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e224130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP323L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e129979\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e168140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR392C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80369\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e153419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eI42V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86910\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2562\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eT112I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSP16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eK160R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe conserved, antigenic, and non-allergenic residues identified in each SARS-CoV-2 protein were subsequently mapped onto corresponding homology models derived from their crystal structures. The functional significance of each residue is detailed below.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eSpike Protein\u003c/strong\u003e:\u003c/h3\u003e\n\u003cp\u003eOur analysis revealed that the Spike (S) protein, which is 1273 amino acids long, contains 49 conserved regions (Table 2, Supplementary Table 1). Of these, 11 were found to be antigenic and non-allergenic peptides (Table 2, Supplementary Table 1, Fig. 2A). Identifying conserved regions within the S protein is pivotal for developing broad-spectrum antivirals and vaccines. Despite the high mutation rate observed in various S protein domains, certain regions remain highly conserved, underscoring their functional importance. The N-terminal conserved domain (NTD) peptide (293\u0026ndash;338) is implicated in host-cell recognition and has been identified as a target for neutralising antibodies (X. Xia, 2021). Mutations in this region can contribute to immune evasion; however, conserved epitopes within the NTD may serve as viable targets for vaccine development (X. Xia, 2021). The S protein\u0026rsquo;s Receptor Binding Domain (RBD) is critical for binding to the ACE2 receptor, facilitating viral entry into host cells (Tian et al., 2021). While the RBD is subject to frequent mutations, specific conserved sequences within this domain are essential for maintaining its structural integrity and function. One of the identified conserved peptides is mapped (510\u0026ndash;519) in the RBD. Notably, the N501Y mutation has strengthened the binding affinity between the spike protein and ACE2 receptor, enhancing viral infectivity (Tian et al., 2021). The H519N mutation significantly decreases SARS-CoV-2 replication in human lung epithelial cells and reduces infectivity in pseudotyped virus (Cereghino et al., 2024). Targeting these conserved regions could lead to the development of broad-spectrum neutralising antibodies (Huan et al., 2024). The fusion peptide of the spike (S) protein plays a critical role in mediating the fusion between the viral and host cell membranes, an essential step in viral entry (Hoffmann et al., 2020). The high conservation of fusion peptide sequences across various coronavirus strains suggests their potential as universal vaccine targets. Peptide-based fusion inhibitors derived from these conserved sequences have demonstrated efficacy against emerging coronaviruses (S. Xia et al., 2014). We have identified conserved regions (693\u0026ndash;700, 705\u0026ndash;746, 748\u0026ndash;763) in this region, suggesting these peptides\u0026rsquo; functional significance. S protein\u0026rsquo;s heptad repeat (HR) regions are integral to the conformational changes required for membrane fusion and viral entry (S. Xia et al., 2019). Mimetic proteins structurally imitating the HR1 region in a trimeric coiled-coil conformation showed potential in inhibiting SARS-CoV-2 infection \u003cem\u003ein vitro\u003c/em\u003e (Zhu et al., 2020). Our analysis revealed three conserved peptides (1021\u0026ndash;1039, 1041\u0026ndash;1069, 1092\u0026ndash;1100) in this region. Targeting these conserved regions within the S protein is a strategic approach to developing effective therapeutics and vaccines across multiple coronavirus strains. Focusing on these regions makes it possible to design interventions that maintain efficacy even as the virus undergoes mutations in other regions.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eEnvelope Protein\u003c/strong\u003e:\u003c/h3\u003e\n\u003cp\u003eThe SARS-CoV-2 envelope (E) protein, comprising 75 amino acids, plays a crucial role in virus assembly, budding, and pathogenesis. The conserved antigenic and non-allergenic sequence (residues 36\u0026ndash;48) is located within the hydrophobic transmembrane domain (TMD) of the E protein, which is essential for anchoring it to the viral envelope (Fig.\u0026nbsp;2B, Table\u0026nbsp;2) (Schoeman \u0026amp; Fielding, 2019). Structural studies have shown that the E protein\u0026apos;s TMD forms a pentameric helical bundle, creating a cation-selective ion channel critical for viral pathogenicity, highlighting its role as a potential target for antiviral drugs (Hong et al., 2020; Schoeman \u0026amp; Fielding, 2019). This underscores the TMD\u0026apos;s potential as a therapeutic intervention target for hindering virus assembly and release. Targeting this region could lead to the design of inhibitors that disrupt E protein function, thereby mitigating SARS-CoV-2 infectivity and pathogenicity.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eMembrane Protein:\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 membrane (M) protein, comprising 222 amino acids, plays a vital role in viral assembly, morphogenesis, and pathogenesis (Z. Zhang et al., 2022). It is the most abundant structural component of the virus and interacts with other essential proteins, such as S and E proteins, to regulate viral budding and genome encapsulation (Z. Zhang et al., 2022). Four of the seven conserved regions observed in the current study were found to be antigenic and non-allergenic (Fig.\u0026nbsp;2C, Table\u0026nbsp;2). The transmembrane location of the conserved region (residues 20\u0026ndash;27) could contribute to its anchoring within the viral envelope and is essential for membrane curvature during viral budding (Neuman et al., 2011). Hydrophobic interactions within this region facilitate lipid bilayer integration, ensuring the stability of the viral structure (Dolan et al., 2022). Another conserved region (residues 40\u0026ndash;62) is known to be critical for protein-protein interactions involved in viral assembly (Z. Zhang et al., 2022). Studies suggest that mutations in this domain disrupt virion formation, producing non-infectious viral particles (Siu et al., 2008). The conserved region (residues 105\u0026ndash;184) is known to interact with the nucleocapsid (N) protein, which plays a key role in encapsulating the viral RNA genome (Siu et al., 2008). Disrupting this region has been shown to impair viral replication and assembly, making it a key target for antiviral drug development (Siu et al., 2008). The host-cell interaction domain region (residues 186\u0026ndash;206) plays a fundamental role in binding to host cell membranes and is involved in the budding process of new virions (Siu et al., 2008). Computational docking studies suggest that peptides mimicking this domain could act as viral assembly inhibitors, preventing new virions from exiting the host cell (Neuman et al., 2011). Given its role in viral structure and function, the M protein is a key therapeutic intervention target. Conserved sequences within the M protein offer promising opportunities for developing antiviral agents that can disrupt viral assembly and budding (Neuman et al., 2011). In addition, targeting the M protein interactions with S and E proteins could prevent the formation of fully functional virions, reducing viral spread within infected individuals (Boson et al., 2021). Moreover, the immunogenic properties of the M protein suggest its potential use in vaccine formulations. The conserved nature of the M protein ensures broad-spectrum vaccine effectiveness, even against emerging SARS-CoV-2 variants.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eNucleocapsid Protein:\u003c/h3\u003e\n\u003cp\u003eThe nucleocapsid (N) protein of SARS-CoV-2, a crucial structural component, is involved in RNA binding, genome packaging, and viral replication (Kang et al., 2020). Comprising 419 amino acids, the N protein exhibited 14 conserved regions, with two areas being antigenic and non-allergenic, underscoring its evolutionary stability across different variants. This conservation suggests a crucial functional significance, making the N protein a prime target for diagnostic and therapeutic applications. Both the conserved antigenic residues (residues 102\u0026ndash;118 and residues 301\u0026ndash;318) (Fig.\u0026nbsp;2D, Table\u0026nbsp;2) are particularly noteworthy due to their potential involvement in RNA binding and protein-protein interactions (Cubuk et al., 2021). The first conserved peptide sequence (residues 102\u0026ndash;118) falls within a highly structured region, which may contribute to the protein\u0026apos;s ability to interact with viral and host components (Cubuk et al., 2021). Similarly, the second conserved peptide sequence (residues 301\u0026ndash;318) is positioned within a flexible domain that may facilitate dynamic conformational changes essential for nucleocapsid function (Cubuk et al., 2021; Dinesh et al., 2020). The high conservation of these regions across SARS-CoV-2 variants suggests their indispensable role in viral assembly and propagation. The N protein is highly immunogenic so that these conserved sequences may serve as potential epitopes for monoclonal antibody development and vaccine design (Dutta et al., 2020). Additionally, the involvement of the identified conserved sequences in viral RNA replication processes suggests that targeting them with small molecules or peptide inhibitors could disrupt viral replication, offering a promising therapeutic avenue (Iserman et al., 2020). The N protein remains a primary target for rapid antigen detection tests in diagnostic applications due to its abundance during infection. Monoclonal antibodies directed against the identified conserved regions could enhance the sensitivity and specificity of lateral flow assays, aiding in early and reliable SARS-CoV-2 detection (Grant et al., 2021). In conclusion, the remarkable conservation of these two regions within the SARS-CoV-2 N protein highlights their functional importance in viral pathogenesis. Future studies should focus on elucidating their structural and functional roles in greater detail, paving the way for improved antiviral strategies and diagnostic tools.\u003c/p\u003e\n\u003ch3\u003eNon-structural protein 1:\u003c/h3\u003e\n\u003cp\u003eNSP-1 is a key virulence factor in suppressing host immune responses and facilitating viral replication (Thoms et al., 2020). Of the seven conserved regions in the NSP-1, two peptides were found to be antigenic and non-allergenic. One of the peptides (residues 50\u0026ndash;59) was mapped onto the surface of the NSP-1 (Fig.\u0026nbsp;3A, Table\u0026nbsp;2). This region (residues 50\u0026ndash;59) within the N-terminal domain (NTD) of SARS-CoV-2 NSP-1 may play a role in host immune suppression and viral replication. Structurally, this region forms part of the \u0026beta;2\u0026ndash;\u0026beta;3 loop, contributing to the electrostatic surface properties of the NTD and stabilising its interactions with host factors (Li et al., 2023). The acidic patch formed by residues E55 and E57 is located adjacent to the \u0026alpha;1-helix and may play a role in the selective binding of viral RNA, while simultaneously inhibiting host mRNA translation (Thoms et al., 2020). Functionally, NSP-1 inhibits host gene expression by blocking translation and suppressing mRNA transport (Schubert et al., 2020). Mutational studies indicated that alterations in the 50\u0026ndash;59 loop impair NSP-1\u0026rsquo;s ability to suppress host defences, leading to attenuation of viral replication (Lapointe et al., 2021). Deletion or mutation of residues in this region has been linked to reduced cytotoxicity and loss of host shutoff function, suggesting its importance in NSP-1\u0026rsquo;s virulence mechanism (Kamitani et al., 2009). Additionally, attenuated viral strains with targeted mutations in this region may serve as candidates for live-attenuated vaccine development (Lapointe et al., 2021). This conserved region presents a promising therapeutic target for antiviral strategies and vaccine design by enabling immune evasion and viral replication.\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eNon-structural protein 3:\u003c/h2\u003e\n \u003cp\u003eThe NSP-3 of SARS-CoV-2 is reported to play a role in viral replication and the modulation of host immune responses (Lei et al., 2018). In our study, NSP-3 is the protein identified with the maximum number of conserved residues with antigenic significance (N\u0026thinsp;=\u0026thinsp;59, N\u0026thinsp;=\u0026thinsp;21, respectively) (Table\u0026nbsp;2, Fig.\u0026nbsp;3B). Other researchers analysed several key amino acid sequences within NSP-3 for their structural and functional significance in viral pathogenesis (Low et al., 2022). The N-terminal region, consisting of residues 1\u0026ndash;8, plays a crucial role in the early stages of viral replication and is also associated with ubiquitin-like domain 1. This domain has been implicated in protein-protein interactions that may modulate host immune responses (Gao et al., 2020). Similarly, residues 96\u0026ndash;107 are located within the SARS-Unique Domain (SUD), a region unique to SARS-related coronaviruses, which interacts with host cell proteins and may influence viral replication efficiency (Lei et al., 2018). The papain-like protease (PLPro) domain, containing residues 356\u0026ndash;363, plays a pivotal role in cleaving the viral polyprotein and has de-ubiquitinating functions that help SARS-CoV-2 evade immune detection (Bailey-Elkin et al., 2017). Structural studies have highlighted the significance of PLPro in counteracting host antiviral responses, making it a key therapeutic target (Shin et al., 2020).\u003c/p\u003e\n \u003cp\u003eAdditionally, the macrodomain 1 of NSP-3, which includes residues 435\u0026ndash;461, has been identified as essential in interfering with the host\u0026apos;s ADP-ribosylation signalling. This domain is vital for viral replication and immune evasion, with structural analyses emphasising its potential as a drug target (Frick et al., 2020). The transmembrane regions of NSP-3, represented by residues 535\u0026ndash;557 and 846\u0026ndash;859, contribute to double-membrane vesicles (DMVs), which provide a protected niche for viral RNA synthesis (Wolff et al., 2020). These domains interact with NSP-4 and NSP-6, highlighting their role in viral replication complex formation. The SUD contains multiple functionally relevant sequences, residues 740\u0026ndash;748 and 750\u0026ndash;770, which are implicated in host-pathogen interactions and may influence the efficiency of viral replication (Tan et al., 2021). Studies suggest that SUD enhances viral pathogenicity through interactions with cellular proteins, further supporting its significance in SARS-CoV-2 infection dynamics. Further downstream, residues 1844\u0026ndash;1866 and 1893\u0026ndash;1945 are located in regions that may contribute to NSP-3\u0026apos;s overall structural stability and interactions with other viral and host components(Lei et al., 2018). Their functional implications warrant further investigation, particularly in viral replication and immune evasion mechanisms. The comprehensive analysis of these NSP-3 sequences provides valuable insights into their functional roles in SARS-CoV-2 pathogenesis. Given their involvement in viral replication, immune modulation, and host interactions, these sequences are potential targets for antiviral drug development. Further structural and biochemical studies must elucidate their full mechanistic contributions and therapeutic potential.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eNon-structural protein 4:\u003c/h2\u003e\n \u003cp\u003eSARS-CoV-2 non-structural protein 4 (NSP-4) is crucial for membrane remodelling during viral replication, interacting with NSP-3 to promote the formation of double-membrane vesicles (DMVs) (Angelini et al., 2013; Oudshoorn et al., 2017). Our analysis revealed 20 conserved residues, with 6 having antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;3C). These six conserved motifs in NSP-4, including residues 61\u0026ndash;94, 97\u0026ndash;111, 129\u0026ndash;136, 138\u0026ndash;145, 253\u0026ndash;263, 387\u0026ndash;400, have been shown to possess functional importance (C. J. Gordon et al., 2020). Structurally, these conserved sequences map to key domains, including luminal loops involved in membrane curvature (61\u0026ndash;145, 253\u0026ndash;263), transmembrane helices forming the DMV-spanning pore (387\u0026ndash;400), and the cytosolic C-terminal tail critical for replication complex assembly (493\u0026ndash;500) (Neuman, 2016; Zimmermann et al., 2023). Notably, N-glycosylation at Asn131 and multiple disulfide bonds within luminal loops suggest an additional layer of regulation influencing NSP-4 stability and interaction with NSP-3 (Oostra et al., 2008). Despite NSP-4\u0026rsquo;s high conservation, the T492I mutation, which emerged in Delta and Omicron variants, enhanced viral replication by increasing 3CLpro processing efficiency (X. Lin et al., 2023). This highlights how even minor mutations in NSP-4 can impact viral fitness. Given its critical role in DMV formation, NSP-4 represents an attractive antiviral target, particularly disrupting the NSP\u0026ndash;3\u0026ndash;NSP\u0026ndash;4 interface or DMV-spanning pore (V\u0026rsquo;kovski et al., 2021). Future studies should explore small molecules or antibodies targeting NSP4, given its low mutation rate compared to other viral proteins, making it a viable broad-spectrum target across coronaviruses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eNon-structural protein 5:\u003c/h2\u003e\n \u003cp\u003eThe NSP-5 main protease (Mpro) of SARS-CoV-2 plays a pivotal role in viral replication by cleaving polyproteins (pp1a/pp1ab) into functional non-structural proteins (L. Zhang et al., 2020). The enzyme is highly conserved, with key sequence segments 36\u0026ndash;74 and 248\u0026ndash;259 demonstrating strong purifying selection across SARS-CoV-2 variants and related coronaviruses (C. J. Gordon et al., 2020). Interestingly, in our study of the 11 conserved residues identified, these two have antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;3D). The 36\u0026ndash;74 region contributes to the catalytic domain (domain I) and harbours His41, a critical residue forming the His41\u0026ndash;Cys145 catalytic dyad essential for substrate cleavage and viral replication (Zhou et al., 2020). This segment also defines the substrate-binding pocket and interacts with known inhibitors such as Paxlovid (nirmatrelvir) (Owen et al., 2021). The 248\u0026ndash;259 region within domain III is crucial for dimerisation, an essential step in NSP-5 activation (Kneller et al., 2020). Disrupting this interface destabilises the enzyme, leading to a loss of function and viral replication inhibition (Su et al., 2022).\u003c/p\u003e\n \u003cp\u003eDespite the emergence of SARS-CoV-2 variants of concern, NSP-5 remains highly conserved, with minimal mutations in its active site or dimerisation interface (Sacco et al., 2020). Mutational studies confirm that substitutions in these regions impair viral fitness, highlighting their functional importance (Jin et al., 2020). Given the stability of these sequences, Mpro remains a prime antiviral target. Covalent inhibitors like nirmatrelvir and peptidomimetics have been designed to irreversibly bind the active site, effectively blocking viral replication (Owen et al., 2021). Additionally, allosteric inhibitors targeting the dimerisation interface have shown promise, offering an alternative antiviral approach (Kubra et al., 2023). Given these segments\u0026rsquo; extreme conservation and functional indispensability, targeting NSP5 with direct-acting antivirals provides a robust therapeutic strategy against SARS-CoV-2 and future coronavirus outbreaks (L. Zhang et al., 2020). Future research should focus on dimer interface inhibitors to complement existing active-site-targeting drugs, ensuring broad-spectrum efficacy against coronaviruses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eNon-structural protein 6:\u003c/h2\u003e\n \u003cp\u003eThe NSP-6 of SARS-CoV-2 promotes the virus\u0026apos;s ability to remodel host cell membranes, facilitating DMVs essential for viral RNA replication. This function is conserved across coronaviruses, underscoring the importance of specific sequences within NSP-6 (Benvenuto et al., 2020). We identified nine conserved residues in this protein, with 4 exhibiting antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;3E). The sequences with residues 12\u0026ndash;23 and residues 25\u0026ndash;34 are located within the transmembrane domains of NSP-6, which are highly conserved among SARS-CoV-2 variants and related coronaviruses, including SARS-CoV and MERS-CoV. This conservation suggests a fundamental role in membrane association and DMV formation (D. E. Gordon et al., 2020a). Similarly, the sequences with residues 169\u0026ndash;179 and residues 222\u0026ndash;231 are preserved across various coronavirus species, indicating their critical role in viral replication (Cottam et al., 2014). The transmembrane regions encompassing residues 12\u0026ndash;34 are integral to NSP-6\u0026rsquo;s ability to anchor to the endoplasmic reticulum membrane. This anchorage is vital for inducing autophagosome formation and subsequent DMV development in viral replication (D. E. Gordon et al., 2020b). The structural conservation of these sequences ensures the integrity required for proper membrane curvature and vesicle formation. Residues 169\u0026ndash;179 and 222\u0026ndash;231 are implicated in protein-protein interactions within the viral replication complex, facilitating the coordination necessary for efficient RNA synthesis (Cottam et al., 2014). Mutations within these conserved regions can significantly impact NSP-6 function. For instance, alterations in the transmembrane domains may disrupt membrane association, hindering DMV formation and attenuating viral replication (Yang et al., 2020). Targeting these conserved sequences with antiviral agents could impair NSP-6\u0026rsquo;s function, offering a potential therapeutic strategy (Kang et al., 2020). Compounds that disrupt NSP-6\u0026rsquo;s membrane interactions or their role in autophagosome formation could effectively inhibit viral replication (Benvenuto et al., 2020). The conserved sequences within NSP-6 are integral to modifying host cell membranes for viral replication. Their preservation across coronavirus species highlights their essential role and presents opportunities for targeted antiviral interventions. Given these sequences\u0026rsquo; high conservation and functional importance, NSP-6 remains a viable antiviral target, particularly for strategies that disrupt its interactions with host cell membranes and interfere with DMV formation (Kang et al., 2020).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eNon-structural protein 12:\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 NSP-12 functions as the RNA-dependent RNA polymerase (RdRp), a crucial enzyme for viral genome replication (Hillen et al., 2020). We identified 31 conserved residues in this protein, with 6 exhibiting antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;4A). These six conserved sequence regions (residues 186\u0026ndash;226, 294\u0026ndash;322, 401\u0026ndash;462, 464\u0026ndash;486, 672\u0026ndash;693, and 720\u0026ndash;738) have been shown to play essential roles in RdRp function, structural stability, and antiviral drug interactions. Residues 186\u0026ndash;226 of NSP-12 facilitate nucleotidyl transfer reactions necessary for RNA synthesis and viral RNA capping (Slanina et al., 2021). Key residues within this region participate in ATP and GTP binding, making it an attractive antiviral target(Subissi et al., 2014) Region 294\u0026ndash;322 contains a highly conserved Cys/His-rich zinc-binding motif stabilising NSP-12 by coordinating Zn\u0026sup2;⁺ ions (Gao et al., 2020). Structural studies indicate that disrupting these zinc-coordinating residues impairs polymerase activity (Q. Wang et al., 2020). Polymerase core regions with residues 401\u0026ndash;486, 672\u0026ndash;693, 720\u0026ndash;738 form key subdomains essential for RNA synthesis. The fingers domain 401\u0026ndash;486 contributes to RNA binding and cofactor interactions, particularly with NSP-8, which enhances processivity (Hillen et al., 2020). The palm domain 672\u0026ndash;693 contains motif B, a flexible loop that adjusts during nucleotide incorporation, facilitating efficient RNA synthesis (Shannon et al., 2020). The 720\u0026ndash;738 region lies adjacent to the active site and stabilises the polymerase\u0026rsquo;s catalytic domain (Yin et al., 2020). The high conservation of these sequences makes them ideal targets for antiviral drugs. Remdesivir, a nucleotide analogue, binds to the polymerase active site and disrupts RNA synthesis by causing delayed chain termination (C. J. Gordon et al., 2020). Structural studies have shown that remdesivir interacts with conserved residues in the fingers and palm domains, stabilising an inactive polymerase complex (Kokic et al., 2021). Molnupiravir and favipiravir also exploit conserved residues to induce lethal mutagenesis (Agostini et al., 2018). These regions remain highly conserved across SARS-CoV, MERS-CoV, and other coronaviruses, indicating strong evolutionary constraints (Pachetti et al., 2020). However, mutations such as P323L (interface domain) and G671S (motif B loop) have emerged in SARS-CoV-2 variants, potentially enhancing viral replication efficiency (S. M. Kim et al., 2023). Nevertheless, remdesivir resistance mutations (e.g., F480L, V557L) remain rare due to the functional constraints on these conserved regions(Agostini et al., 2018) Overall, the conserved sequences in NSP-12 are essential for viral replication and represent key targets for developing antiviral drugs. Their limited mutational tolerance reinforces their potential as drug-binding sites, highlighting the importance of continued structural and functional studies to optimise therapeutic strategies.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eNon-structural protein 13:\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 NSP-13 is a highly conserved helicase that plays a critical role in viral replication by unwinding RNA and hydrolysing ATP (Jia et al., 2019). We identified 24 conserved residues in this protein, with 6 exhibiting antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;4B). These conserved sequences in NSP-13, including residues 1\u0026ndash;17, 54\u0026ndash;76, 141\u0026ndash;153, 414\u0026ndash;430, 432\u0026ndash;443, and 554\u0026ndash;575, correspond to essential functional motifs required for enzymatic activity, structural integrity, and interaction with other viral components. The zinc-binding domain (ZBD), comprising the 1\u0026ndash;17 and 54\u0026ndash;76 sequences, coordinates Zn\u0026sup2;⁺ or Fe\u0026ndash;S clusters necessary for NSP-13 stability and RNA unwinding. Studies have shown that mutations in these cysteine-rich motifs disrupt metal coordination and abolish enzymatic function (Maio et al., 2024). The stalk domain (141\u0026ndash;153) supports the helicase core and facilitates interactions with NSP-12 and NSP-8 in the replication-transcription complex (J. Chen et al., 2020). The helicase core, including 414\u0026ndash;430 (RecA1 domain), 432\u0026ndash;443 (RecA2 domain), and 554\u0026ndash;575 (motif VI), contains essential ATP-binding and RNA-binding motifs. Motif VI, particularly Arg567 within the 554\u0026ndash;575 segment, functions as an \u0026quot;arginine finger,\u0026quot; crucial for ATP hydrolysis and energy transduction (Hao et al., 2017). Structural studies have revealed that these conserved sequences undergo conformational changes upon ATP binding, facilitating RNA unwinding (Jia et al., 2019). NSP13 is a promising antiviral target due to its high conservation across coronaviruses. Small-molecule inhibitors such as bismuth complexes, flavonoids (myricetin, scutellarein), and SSYA10-001 have been shown to target these conserved motifs, disrupting ATPase and helicase activity (Adedeji et al., 2012). Additionally, the ZBD is a druggable site, as bismuth-based compounds destabilise its zinc-finger motifs, effectively inhibiting the helicase (Shu et al., 2020). Comparative sequence analysis across SARS-CoV, MERS-CoV, and other coronaviruses highlights the strong evolutionary conservation of these motifs, underscoring their indispensable role in viral replication, making them attractive targets for therapeutics and vaccines.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eNon-structural protein 14:\u003c/h2\u003e\n \u003cp\u003eThe NSP-14 of SARS-CoV-2 is integral to the virus\u0026apos;s replication fidelity and immune evasion strategies (Y. Chen et al., 2009; Ogando et al., 2020). Our sequence analysis has identified 22 conserved regions within NSP-14, including six antigenic residues: 32\u0026ndash;41, 50\u0026ndash;66, 126\u0026ndash;139, 145\u0026ndash;156, 223\u0026ndash;249, and 361\u0026ndash;370 (Table\u0026nbsp;2, Fig.\u0026nbsp;4C). The conservation of these sequences across various SARS-CoV-2 isolates suggests their critical role in maintaining the structural integrity and enzymatic functions of NSP-14 (Saikatendu et al., 2005). Notably, NSP-14 of SARS-CoV and SARS-CoV-2 share over 95% amino acid sequence similarity, underscoring their evolutionary conservation and potential as a therapeutic target (Robson et al., 2020). The ExoN activity of NSP-14 is essential for correcting errors during RNA synthesis, thereby reducing the mutation rate and contributing to the stability of the viral genome (Bouvet et al., 2010). Additionally, the N7-MTase domain\u0026apos;s role in mRNA capping protects viral RNA from host immune responses, facilitates efficient translation, and ensures viral proliferation (S. Lin et al., 2020). The crystal structure of SARS-CoV-2 NSP-14 demonstrates that the ExoN domain\u0026apos;s enzymatic activity is metal ion-dependent, preferably utilising Mg\u0026sup2;⁺, and that the N7-MTase domain harbours a conserved DxG motif for S-adenosyl-L-methionine binding, characteristic of coronaviruses (Saikatendu et al., 2005).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eNon-structural protein 15:\u003c/h2\u003e\n \u003cp\u003eThe SARS-CoV-2 NSP-15 is an endoribonuclease highly conserved among coronaviruses and plays a crucial role in viral RNA processing and immune evasion (Y. Kim et al., 2020). It cleaves viral RNA at uridine sites, preventing the accumulation of immunostimulatory dsRNA, thereby helping the virus evade host immune responses (X. Deng et al., 2017). We identified 14 conserved residues in this protein, with 2 exhibiting antigenic potential (Table\u0026nbsp;2, Fig.\u0026nbsp;4D). The conserved residues 1\u0026ndash;9 and 321\u0026ndash;336 are functionally significant as they contribute to NSP-15\u0026apos;s hexameric structure and catalytic activity, making them potential antiviral drug targets ((Saramago et al., 2022). The peptide with residues 1\u0026ndash;9 at the N-terminus is part of the oligomerisation domain, essential for forming the active hexameric complex (Y. Kim et al., 2020). Mutations in this region impair hexamerization and lead to loss of enzymatic activity, suggesting its importance in structural integrity (Ivanov et al., 2004) The conservation of this motif across coronaviruses highlights its critical role in viral replication and potential as a drug target (X. Deng et al., 2017). The peptide with residues 321\u0026ndash;336 in the C-terminal region is located near the active site of NSP-15 and is highly conserved across beta-coronaviruses (Saramago et al., 2022). It contributes to substrate recognition and enzymatic function, ensuring efficient RNA cleavage (Saramago et al., 2022). Structural studies show that this sequence forms part of the uridine-binding pocket, crucial for its RNA endonuclease activity (Hackbart et al., 2020). Mutations in this region disrupt catalytic efficiency, accumulating dsRNA and increasing host immune activation (X. Deng et al., 2017). Viruses lacking a functional NSP-15 enzyme show reduced replication efficiency and are more susceptible to host immune responses. The conserved residues 1\u0026ndash;9 and 321\u0026ndash;336 in SARS-CoV-2 NSP-15 are structurally and functionally crucial. They contribute to enzyme oligomerisation, substrate recognition, and RNA processing, ensuring viral replication and immune evasion. Their high conservation makes them ideal targets for antiviral drug development.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings provide insights into the stability and evolutionary conservation of SARS-CoV-2 protein sequences, suggesting their probable crucial role in developing robust detection assays and targeted therapeutic and preventive interventions. These conserved sequences within SARS-CoV-2 are integral for viral replication and immune evasion. Mutations within these sequences are rare due to their functional constraints, and the reports of experimental mutagenesis studies confirm that disruptions in these regions would lead to loss of viral replication efficiency, making them attractive therapeutic and preventive targets for broad-spectrum SARS-CoV-2 variants. Future research may focus on developing inhibitors that exploit these conserved sites to disrupt viral replication, effectively providing effective therapeutic and prophylactic strategies against SARS-CoV-2 and related coronaviruses.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e1. SARS-CoV-2: Severe acute respiratory syndrome-coronavirus-2\u003c/p\u003e\n\u003cp\u003e2. COVID-199: Corona virus disease-19\u003c/p\u003e\n\u003cp\u003e3. S protein: Spike protein\u003c/p\u003e\n\u003cp\u003e4. RBD: Ribosome binding domain\u003c/p\u003e\n\u003cp\u003e5. HR: Heptad repeat\u003c/p\u003e\n\u003cp\u003e6. E protein: Envelope protein\u003c/p\u003e\n\u003cp\u003e7. TMD: Trans membrane domain\u003c/p\u003e\n\u003cp\u003e8. M protein: Membrane protein\u003c/p\u003e\n\u003cp\u003e9: NSP: Non-structural protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and material: The datasets used/analysed during the current study are available in the NISAID repository, https://gisaid.org\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eFunding: No funding was received for this study\u003c/p\u003e\n\u003cp\u003eAuthors’ contributions: \u003cem\u003eRM, KP, JS, and SR contributed to the study conception and design. JS, SR, ST, BRN, ARA, SKP, AV, JP, VK, MMA, MM, MY, and PKM performed material preparation and data collection. RM, KP, PSD, and AK analysed the data. The first draft of the manuscript was written by RM, KP, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAcknowledgements: The authors would like to acknowledge Osmania University, and PVNRGVU, Hyderabad, for providing resources to conduct the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdedeji, A. O., Singh, K., Calcaterra, N. E., DeDiego, M. L., Enjuanes, L., Weiss, S., \u0026amp; Sarafianos, S. G. (2012). Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase. Antimicrobial Agents and Chemotherapy, 56(9), 4718\u0026ndash;4728. https://doi.org/10.1128/AAC.00957-12,\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgostini, M. L., Andres, E. L., Sims, A. C., Graham, R. L., Sheahan, T. P., Lu, X., Smith, E. C., Case, J. 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Nature Communications, 14(1). https://doi.org/10.1038/S41467-023-43666-5,\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SARS-CoV-2, COVID-19, Structural proteins, Non-structural proteins","lastPublishedDoi":"10.21203/rs.3.rs-6798845/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6798845/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe ongoing evolution of SARS-CoV-2, especially the emergence of heavily mutated variants like Omicron and its sub-lineages, has resulted in antigenic drift that diminishes the effectiveness of current first-generation vaccines, diagnostic tests, and treatments. This study employed a comprehensive immuno-informatics approach to identify highly conserved protein sequences from SARS-CoV-2 isolates reported in India. 1,33,154 complete protein sequences retrieved from the GISAID database between September 2021 and March 2023 were analysed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The analysis revealed a total of 62,94,995 mutations, which include 66,861 unique mutations. Sequences comprising at least eight consecutive amino acids with mutation frequencies below 0.1% were considered conserved regions. This analysis identified 270 conserved sequences across both structural and non-structural proteins. Of these, 73 sequences were found to be antigenic and non-allergenic and were mapped onto their respective crystal structure of proteins to evaluate their functional relevance. Many conserved sequences overlapped with the known functionally significant epitopes conserved across SARS-CoV-2 variants, underscoring their importance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The identified conserved sequences offer valuable targets for developing variant-resilient peptide-based diagnostics, monoclonal antibody therapeutics, and multi-epitope peptide vaccines. This study provides a curated collection of conserved SARS-CoV-2 protein regions identified from Indian clinical isolates and emphasises their potential for diagnostic and therapeutic applications. These findings may contribute to developing universal, variant-proof strategies for SARS-CoV-2 detection, prevention, and treatment.\u003c/p\u003e","manuscriptTitle":"Conserved SARS-CoV-2 Viral Peptides as Potential Prophylactic and Therapeutic Targets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 09:35:12","doi":"10.21203/rs.3.rs-6798845/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e152f55-d3f8-42eb-a605-6095b69b1fbf","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-26T14:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 09:35:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6798845","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6798845","identity":"rs-6798845","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}