The COVID-19 pandemic continues to have a devastating impact worldwide, with over 776 million cumulative cases and more than 7 million cumulative deaths, according to the World Health Organization as of September 2024 (WHO. Number of COVID-19 cases reported to WHO (cumulative total)., 2024). A variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Omicron (B.1.1.529), was first detected in specimens from South Africa on 11 November 2021 and was declared a variant of concern (VOC). The Omicron variant has numerous mutations in its surface glycoprotein (spike protein, S protein), the antigenic target of vaccine-elicited antibodies (Wilhelm, 2021, Chen, 2022). The receptor-binding domain (RBD), which is a primary target for neutralizing antibodies (NAbs), has almost 15 mutations (Harvey, 2021). Several spike protein mutations observed in Omicron were previously reported in variants of concern (VOCs) of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, as well as in variants of interest (Karam, 2020) of, for example, Kappacoronavirus, Zetacoronavirus, Lambdacoronavirus, and Mucoronavirus (Wilhelm, 2021).

SARS-CoV-2 is an enveloped virus with linear positive-sense single-stranded RNA (+ssRNA) as its genetic material (Lai, 2020, Chen et al., 2020). SARS-CoV-2 has a genome of 29,881 bases that encodes at least four viral structural proteins: spike glycoprotein (S), membrane protein (M), an envelope protein (E), and nucleocapsid protein (N). The S protein mediates the viral entrance of SARS-CoV-2 into host cells by binding to the host receptor angiotensin-converting enzyme 2 (ACE2) through the S1 subunit's receptor-binding domain (RBD). Following the S1-ACE2 interaction, the cellular protease 'transmembrane protease serine S1 member 2′ (TMPRSS2) cleaves the S1-S2 fusion peptide. The S2 subunit fuses the viral and host membranes (Luan, 2020, Hoffmann, 2020). However, one study proposed that the cleavage of a furin-dependent furin cleavage site in the S protein occurs before membrane fusion (Coutard, 2020). TMPRSS2 is found only in the lungs and gastrointestinal tract cells, whereas ACE2 is found in cells of other organs and tissues, such as the liver, heart, vascular endothelium, testes, and kidneys (Sakamoto, 2021). The scientific community widely considers the S protein a prime target for producing antibodies, entry inhibitors, and vaccines against SARS-CoV-2 because it plays an essential role in viral fusion and entry (Sadat, 2021, Bhattacharya, 2022, Walls, 2020, Du, 2009, Beniac, 2006). Hence, we used the S protein as a target for developing an epitope vaccine candidate in current research.

Traditional vaccines, such as live attenuated, inactivated pathogen, and subunit vaccines, provide long-term protection against infectious illnesses (Tandrup Schmidt, 2016, Plotkin, 2009). However, systems struggle to match the demand for more rapid development and large-scale production. Moreover, immunogenicity indices for peptide-based vaccines have also been lower (Li, 2014). Meanwhile, the rapidly emerging field of epitope therapies can be a powerful platform to address these issues because of the associated safety, comparatively low production cost, rapid development capabilities, and improved efficacy (Dey, 2023, Mahapatra, 2022, Nguyen et al., 2022). Epitope vaccines elicited more robust immune responses than complete protein vaccines (Kao and Hodges, 2009). In addition, effective adjuvants, nanoparticle delivery methods, and immunogenic carrier proteins could be incorporated into epitope vaccine formulations to improve immunogenicity (Lei, 2019, Kelly et al., 2019). A multi-epitope vaccine (MEV) produces immunological responses based on small immunogenic sequences rather than significant proteins or whole-genome recombinant vaccines (Mahapatra, 2023, Dey, 2023). Besides, based on the following evidence, MEVs are better than single-epitope vaccines. First, major histocompatibility complex (MHC) class I and MHC class II epitopes can be recognized from T-cell subsets with the help of T-cell receptors (TCRs). Secondly, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and linear B lymphocyte (LBL) epitopes, when overlapped, cause the activation of humoral-cellular immune responses. Third, the vaccine ensures a long-term and more effective immune response once joined with an adjuvant. Lastly, the limitations due to in vitro antigen expression complications and the culturing of the pathogens are reduced (Zhu, 2014, Saadi et al., 2017, Mahmoodi, 2016, Lu, 2017, Jiang, 2017). The vaccines designed by this approach have shown in vivo efficacy with protective immunity (Zhou, 2009, Guo, 2013, Cao, 2017) and entered phase I clinical trials, representing the initial stage of human testing for safety and dosage determination (Zhu, 2014, Toledo, 2001, Slingluff, 2013, Lennerz, 2014).

Therefore, this study aims to design a MEV incorporating CTL, HTL, and LBL epitopes derived from the SARS-CoV-2 spike glycoprotein using immunoinformatics approaches. By including these epitopes, the vaccine is designed to elicit both humoral and cellular immune responses. CTLs play a critical role in identifying and eliminating virus-infected cells, while HTLs provide essential cytokine support to enhance B-cell antibody production and cytotoxic T-cell responses. The overlap between CTL, HTL, and B-cell epitopes is expected to trigger a robust immune system activation, promoting the development of memory cells for long-term protection against reinfection. Additionally, the vaccine integrates CD40 ligand (CD40L) as an adjuvant, which enhances the immune response by activating antigen-presenting cells (APCs) such as dendritic cells and macrophages. This activation is crucial as CD40L serves as a co-stimulatory molecule that binds to CD40 on APCs, thereby amplifying the vaccine's overall effectiveness. The binding of CD40L to CD40 on APCs enhances their ability to prime T cells and activates B cells, which are essential for generating a robust immune response (Palumbo et al., 2011, Liu, 2008, Coler, 2015). As a result, this multi-faceted approach is anticipated to generate a robust immune response, offering long-term protection against the Omicron variant.

In summary, by integrating immunoinformatics and computational methods, this study provides valuable insights into the development of next-generation COVID-19 vaccines. The MEV design proposed here has the potential to make significant contributions to ongoing global vaccination efforts, particularly in addressing emerging and future variants of concern. The use of immunoinformatics allows for rapid epitope identification and vaccine optimization, paving the way for efficient and adaptable solutions to combat evolving viral threats.