COVID-19 (coronavirus disease-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan City, China, in late 2019 when unexplained cases of pneumonia were reported. The new strain of this virus was not spotted earlier in humans. In March 2020, the World Health Organization (WHO) proclaimed the novel coronavirus outbreak as a global pandemic (Abd El-Aziz and Stockand, 2020). The virus infected more than 31.6 million people across the globe, leading to more than 971,000 (3.07%) mortalities in 220 countries and territories after 6 months of its outbreak (Weber et al., 2020). Despite strict global constraints and quarantine efforts, the incidence of COVID-19 continues to rise. As of 27 May 2022, 525,467,084 confirmed casesCOVID-19 and 6,285,171 deaths have been reported by the WHO. The CoVs are classified into Alphacoronavirus (αCoVs), Betacoronavirus (βCoVs), Deltacoronaavirus (δCoVs), and Gammacoronavirus (γCoVs). The structural proteins of the coronavirus are spike protein (S), membrane protein (M), nucleocapsid protein (N), and envelope protein (E) (Satarker and Nampoothiri, 2020). The S-protein exhibits different degrees of conservation across the Coronaviridae family. It is a type-I transmembrane N-linked glycosylated protein (Yadav et al., 2021). It binds to the cellular membrane ACE2 receptor (Pohl et al., 2021) that permits the entry of the virus into the host cell and triggers a viral–host membrane fusion (Jaimes et al., 2020). The host cell proteases cleave the S-protein. It does so either following the attachment of virions to the host cell membrane or during virion maturation and exit, which is crucial for the S-protein's fusion function (Ou et al., 2020). The receptor-binding region (RBD) identifies the host ACE2 receptor. Thus, RBD is a crucial factor of virus–receptor interaction that considers host selectivity, virus tropism, and infectivity (Su et al., 2020). In late 2020, the emergence of variants posed an increased risk of transmission to global public health, leading to the classification of variants into variants of interest (VOIs) and variants of concern (VOCs) aligning them for global monitoring and research. Variants of concern include Alpha, Beta, Delta, Gamma, and Omicron. Variants of interest include Kappa, Lambda, Iota, and Epsilon. Several antiviral drugs and treatment agents such as favipiravir, hydroxychloroquine, corticosteroids, chloroquine, monoclonal antibodies, and convalescent plasma have been assessed for treating COVID-19. However, they have not been found to be effective (Gavriatopoulou et al., 2021).

Contrarily, vaccines have so far proven to be the best agents in preventing COVID-19. Vaccines consist of macromolecules from microorganisms or attenuated and killed whole microorganisms that are used to induce an immune response. Moreover, DNA vaccines contain DNA that codes for a protein to induce an immune response. At present, the availability of genomic and proteomic data, advanced software, and immunological datasets considerably assists researchers in detecting the potent epitopes from antigens that can be used to develop active peptide-based vaccines (De Gregorio and Rappuoli, 2012; Kalita et al., 2019). Fragments of antigenic proteins of pathogens are used in peptide-based vaccines to elicit natural defenses against the target pathogen (Saadi et al., 2017). In recent studies, multi-epitope-based peptide (MEBP) vaccine constructs have been designed against different viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) (Tahir et al., 2019), Respiratory syncytial virus (RSV) (Tahir ul et al., 2020), the Ebola virus (Ahmad et al., 2019), the Zika virus (Shahid et al., 2020), Hepatitis C Virus (HCV) (Ikram et al., 2018), HIV (Pandey et al., 2018), the BK virus (Kesherwani and Tarang, 2019), and norovirus (Azim et al., 2019), and promising results have been obtained. In comparison with single-epitope vaccines or conventional vaccines, MEBP vaccines are simple in construction, cost-efficient, time-saving, and stable, and they also have selectivity properties. MEBP vaccines concurrently induce a consequential humoral and cell-mediated immune response because of the existence of dual-purpose epitopes. Therefore, nowadays, MEBP vaccines are preferred, and various clinical trials have been carried out.

A life-threatening viral disease called COVID-19 caused a global pandemic in early 2020 due to its highly evolving nature and transmission rate. Numerous drugs and treatments were available, but none of them were established as effective in suppressing it. Vaccines are a single effective tool that can be used to fight against this deadly infection. The typical approach to vaccine development involves problems associated with its efficiency and expression. To address these problems, the latest approach of an “epitope-based vaccine” is promising compared to using the entire protein used as a vaccine. The accessibility of advanced computational tools and proteome information concerning an organism simplifies the creation of a subunit-based vaccine, which is antigenic and capable of eliciting an immune response. Immunoinformatics plays a significant role in the development of a stable, effective, and safe vaccine for human administration.

The genome of SARS-CoV-2 is 29,903 bp long with a linear ss-RNA. This study targeted the surface glycoprotein, the spike protein of SARS-CoV-2 (CDS: 21563.25384), which is significant for viral infections in humans due to its high antigenicity. Various computational tools were used for selecting the antigenic region of the protein to develop a vaccine. Before selecting the epitopes, mutations of SARS-CoV-2 (Delta, Kappa, Lambda, and Iota) obtained from the co-variants online tool were introduced into the spike reference protein sequence to understand the extent of immune response that can be caused in the host body. The SNQVAVLYQGVNCTE epitope contains the mutation D614G, which is a common mutation in all variants that can produce a good immune response. T-cell and B-cell epitopes of the S-protein containing the mutations of the variants were used for vaccine construction. The B-cell and T-cell epitopes were selected using the ABCpred tool and the IEDB online server, respectively, and their antigenicity and allergenicity were tested using VaxiJen v2.0 and AllerTOP v2.0, respectively.

Variations between Omicron and Delta variants have been utilized to design multi-epitope-based vaccines (Dawood, 2022). In this study, variations among four variants, Delta, Kappa, Lambda, and Iota, were utilized to design a multi-epitope-based vaccine. The interaction of the vaccine construct designed in this study with TLR showed better results with TLR 9. Contrary to this finding, a vaccine construct based on the Omicron and Delta variants showed better results with TLR 3 (Dawood, 2022).

The IEDB population coverage tool results showed that 79.78% of the world population was covered by the vaccine construct, based on its interactions with multiple HLA alleles. Adjuvant, B-cell, and T-cell epitopes, as well as a Histidine tag, were built into the vaccine and held together by linkers (EAAAK, GPGPG, and AAY). There are three B-cell epitopes, three MHC Class-I binding epitopes, and three MHC Class-II binding epitopes. The T-cell epitopes were selected based on their binding affinity for MHC-I and MHC-II molecules. Adjuvant β-defensin was attached at the C-terminal to protect the vaccine from degradation, to enable efficient binding to the TLR molecules, and to ensure that the vaccine acts as an agonist and enhances the body's natural defense. The adjuvant β-defensin was linked with B-cell epitopes using the EAAAK linker. The GPGPG linker was used to link B-cell epitopes and cytotoxic T-cell epitopes with MHC Class-I binding epitopes using the AAY linker. To facilitate the cloning of the vaccine construct into vectors, the His tag was attached at the N-terminal of the vaccine construct. The final vaccine construct was both non-allergenic and antigenic and proved to be an effective candidate for providing immunity. The vaccine construct was 228 amino acids long, with a molecular weight of 23943.73 Da. The isoelectric point for the vaccine construct suggested that it is basic. Extinction coefficient refers to the absorption of light in a medium.

The average extinction coefficient was measured to be 17732.5. The extinction coefficient can be used for further spectral UV analysis studies on the vaccine construct. The GRAVY index and the instability index specified the stability of the vaccine construct. The GRAVY index of the vaccine construct was −0.042. Proteins with a GRAVY index <0 are more likely to be hydrophilic (Atapour et al., 2020). An instability index of <40 is considered a stable protein. The instability index of the vaccine construct was 36.80, indicating its stability. The relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine) is referred to as the aliphatic index. It enables further studies on the thermostability of globular proteins. The aliphatic index in the range of 66.5–84.33 indicates the thermal stability of the protein (Panda and Chandra, 2012).

The aliphatic index of the vaccine construct was 73.07, indicating a thermally stable construct. The short half-life of the peptide suggested that it can remain viable for an adequate span of time to generate a potent immune response. The RaptorX server was used to assess the 3D structure of the vaccine construct, and it was refined with the Swiss-PdbViewer Web server for energy minimization. The ProSA online server was used to evaluate the overall and local quality of the refined model and to predict the free energy graphically. The overall model quality indicates the quality of models from distinct sources, such as NMR and X-ray. The local model quality gives energies as a function of amino acid sequence positions. Single-residue energies contain extensive fluctuations and have confined values for model evaluation; therefore, a window size of 40 residue fragments was chosen. Free energy representation indicates the increasing order of residue energy from blue to red.

After refinement and validation of the vaccine model, the quality of the predicted model was observed to be standard at 85%, as more residues were found in favored regions. Codon adaptation showed an increase in the expression of the MEBP vaccine insert in strain K12 of E. coli. The protein–peptide interaction showed the significant binding affinity of the MEBP vaccine construct with TLR 3 and TLR 9. This indicates that the immune system will be able to recognize the molecular patterns of the virus and trigger an immune reaction. Secondary structure scrutinization of docked complexes using PDBsum helped obtain the number of turns, helices, and strands present in individual TLRs and vaccine construct chains, indicating the flexibility of the vaccine complex. It also predicted the protein–protein interface and the bonds formed in the interface region. This included information about the area occupied by the interface and the residue interactions with various bonds (H-bonds, disulfide bonds, non-bonded contacts, and salt bridges) between the TLR chain and the vaccine chain, indicating the stability and extent of interaction within the docked complex.

Molecular dynamics simulation: MD simulation was performed for the TLR 3-vaccine complex and the TLR 9-vaccine complex to comprehend the structural consequence of the MEBP vaccine construct under the simulation condition. Five parameters, RMSD, radius of gyration, H-bonds, RMSF, and PCA, were evaluated. RMSD is normally used to indicate the structure approaching an equilibrium state. It is a measure of distance; the higher the RMSD value, the lower the stability of the complex and vice versa. The trajectory in the graph indicated structural stability at ~50 ns. The RMSF indicated the variance of atoms about its normal position. This helped us understand the flexibility of different regions of the complex. According to the results obtained, the TLR9-vaccine complex showed less flexibility than the TLR3-vaccine complex. This indicates that the TLR9-vaccine complex is more stable than the TLR3-vaccine complex. The radius of gyration can be used to understand the shape of the molecule at any instant in time. A stable Rg value of the TLR9-vaccine complex indicated a stable fold. H-bonding is a property that calculates the total number of hydrogen bonds, including those within the peptide or between the peptide and the surrounding solvent. The presence or absence of an H-bond is traced from the distance between a donor-H-acceptor angle and the donor-H-acceptor pair. The TLR 9-vaccine complex showed a higher number of hydrogen bonds, indicating higher stability. PCA gives the fluctuations in the total number of amino acid residues, where the lower the fluctuation, the more stable and compact the protein structure. Hence, the TLR 9-vaccine complex is more stable.

Immune simulation of the MEBP vaccine construct showed that it can trigger an outstanding immune reaction by producing pathogens and neutralizing antibodies and other immune cells after administration. For a maximum antigen count per milliliter (700,000/ml), the antibody concentration in terms of IgM (2,300/ml), IgG1 (1,000/ml), IgG2 (0/ml), IgG1+IgG2 (up to 3,000/ml), and IgM+IgG (3,200/ml) indicated a good response. It can also be concluded that both humoral and cell-mediated immune responses were triggered. Computational and immunoinformatics tool studies verified that the vaccine construct is stable, highly effective, and safe as a target for an advanced vaccine against SARS-CoV-2. Furthermore, the accuracy and effectiveness of vaccines can be understood through in-vitro studies.

In our study, we utilized reverse vaccinology and immunoinformatics approaches to design a multi-epitope peptide-based vaccine construct for immunization against the emerging variants of SARS-CoV-2, such as Alpha, Beta, Delta, Gamma, all Omicron variants, Kappa, Lambda, Iota, and Epsilon. This multi-epitope-based vaccine construct was found to have the potential to generate both B-cell and T-cell-mediated immune responses against COVID-19, thereby creating a strong resistance inside the body. Various online tools, servers, and in-silico approaches were used to predict epitopes, antigenicity, allergenicity, and population coverage, to analyze the secondary structure, generate a 3D model, and ensure its validation and simulation in the human body. The molecular interaction between TLR molecules and the vaccine construct was studied.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

KK designed the study and wrote the protocol and the first draft of the manuscript. KK, YK, SB, SS, TM, KS, BA, MS, DR, SMS, and MM conceived the study and contributed to the writing and editing of the manuscript. All authors read and approved the final manuscript.