1. IntroductionCrimean–Congo haemorrhagic fever (CCHF) is caused by the human pathogenic agent Crimean–Congo haemorrhagic fever virus (CCHFV). It is known to have affected more than 30 countries and discrete areas around the world [1]. CCHFV belongs to the genus Nairovirus of the family Bunyaviridae; the nairoviruses are mainly tick-borne viruses [2]. The RNA genome of the Bunyaviridae family members harbours three negative-sense segments, S (small), M (medium), and L (large), which minimally code for the virus nucleocapsid (N), glycoprotein precursor (GPC), and polymerase (L) proteins, respectively [3]. Two structural glycoproteins (i.e., G1 and G2, also known as GN and GC, respectively) are encoded by the M segment [4,5].The virus glycoproteins play a salient role in the natural tick–vertebrate cycle of CCHFV and are also responsible for high pathogenicity in humans. Identification of a highly variable mucin-like region at the amino terminus of the CCHFV glycoprotein precursor represents a distinct feature of nairoviruses belonging to the family Bunyaviridae [4]. As surface glycoproteins are important elements facilitating the interaction of CCHFV with its receptor, neutralizing antibodies are engendered against these proteins, making them an attractive target for designing potential vaccines against CCHFV [6]. Previous studies have reported that structural glycoproteins-based vaccines for CCHFV resulted in varying degrees of protection from viral infection in in vivo models [6,7,8,9].CCHFV was initially reported in the 1940s among agricultural workers of the Crimean peninsula, where more than 200 cases of severe haemorrhagic fever were found [10]. Due to the severity of this disease, the fatality rate can rise to 30%. Its broad distribution around the world includes a significant part of the Middle East, Africa, Asia, and regions of Eastern Europe [11,12,13]. No vaccine is available to combat CCHFV infection in humans or animals [14]. The disease progresses rapidly in humans and results in acute febrile illness linked with petechiae, ecchymosis, disseminated intravascular coagulation, and multiple-organ failure [11]. CCHFV spreads throughout the body during the course of the disease, indicated by its detection in the spleen, lung, heart, and intestinal tissues in fatal cases [15]. Among the main cellular targets of CCHFV, infection of monocyte-derived macrophages, endothelial cells, and dendritic cells has been confirmed in vitro [16,17,18]. Owing to the error-prone polymerase, random mutations are incorporated into the single-stranded-RNA-containing genome of CCHFV. This, coupled with a high rate of recombination in CCHFV RNA, makes the development of a vaccine or antiviral drug against CCHFV onerous [19]. The efficacy of Ribavirin against CCHFV has been indicated in vitro and in animal models, but the clinical benefits of Ribavirin remain unwarranted [20].Based on phylogenetic analysis, CCHFV is subdivided into seven distinct genetic classes: Africa 1–3, Asia 1–2, and Europe 1–2, which correlate to the geographic origin [3]. Since the detection of the first case in 1976, CCHF infection has been endemic in Pakistan. A total of 688 confirmed cases were reported between 2012–2022, with most cases reported from Pakistan’s Khyber Pakhtunkhwa and Baluchistan provinces. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan [21,22].A multi-epitope vaccine composed of a collection of epitopes is a viable strategy for preventing and treating viral infection [23]. Besides, multi-epitope vaccines have the benefit of inducing humoral, innate, and cellular immune responses at the same time when compared to monovalent vaccines [24]. Traditional vaccine development procedures are time-consuming and labour-intensive [25]. Immunoinformatics-based tools, on the other hand, can evaluate the host immune response to present an alternative way for developing cost-effective vaccines against diseases because predictions can reduce the number of in vitro experiments required [26,27]. In addition, vaccines based on structural and non-structural proteins have been shown to induce protective immune responses against pathogens [28,29]. Herein, we used several in silico and immunoinformatics-based methods to predict immunodominant B-cell and T-cell epitopes from G1 and G2 of the Asia-1 Genotype of CCHFV and design a multi-epitope vaccine capable of protecting against the viral infection. 4. DiscussionEpitope-based vaccine designing has gained considerable attention owing to its promising features over traditional vaccination. Unlike the single epitope or conventional vaccine, the multi-epitope vaccine benefits from including several MHC-restricted epitopes that T-cell receptors can recognize from various T-cell subsets. They can simultaneously elicit humoral and cellular immune responses due to the inclusion of CTL, HTL, and B-cell epitopes. Besides, the multi-epitope vaccine contains adjuvant substances that can enhance immunogenicity and minimize the use of undesirable substances which can otherwise cause abnormal immunological reactions [23]. Previously, several immunoinformatics-guided studies have designed the multi-epitope vaccine against pathogenic viruses, such as Cytomegalovirus, Dengue, Ebola, Hepatitis C, MERS-CoV, SARS-CoV-2, Zika, etc. [48,49,50]. Experimental approaches have validated the efficacy of in silico constructed multi-epitope vaccine against Mycobacterium tuberculosis [51] and SARS-CoV-2 [52]. Also, multi-epitope vaccine designed using a similar strategy has been shown to exhibit protective effects in vivo [53,54], and several of these vaccines have progressed to the clinical trial stage [55,56,57]. Considering the significant health burden associated with CCHFV (Asia-1 genotype), designing a multi-epitope vaccine against the virus could be desirable. The current study aimed to formulate a multi-epitope vaccine using the immunoinformatics-guided approaches capable of eliciting a robust immune response against the CCHFV (Asia-1 genotype) infection in humans.Typically, pathogens’ surface proteins are more likely to interact with the host’s immune system and trigger an immunological response [58]. Here, we screened the surface glycoproteins (G1 and G2) of CCHFV to predict the immunodominant B-cell and CTL epitopes. This feature is crucial for the vaccine design, as B-cells are associated with antibodies production and CTLs show a major cytotoxic activity against cells infected with intracellular microbes [59]. Besides, the HTL epitopes can be mapped from the structural glycoproteins of CCHFV (Asia-1 genotype) for novel multi-epitope-based vaccine designing. Antigenicity prediction indicated the immunogenic potential of epitope as probable antigen and non-allergenic character labelled them safe from causing harmful allergen reactions in humans. The selected CTL epitopes showed high affinity for HLA-A*02:01 and HLA-B*44:02 alleles, which are reported to have an overall average frequency of 15.28% and 21.62%, respectively [60] around the world. Linking the finalized six CTL and five B-cell epitopes and fusing with other chimeric vaccine components were done using multiple linkers. The main advantage of using linkers in a multi-epitope vaccine is that they avert the formation of junctional immunity and assist in the processing and presentation of antigens [61]. Following the literature [52,62,63,64], several linkers (EAAAK, GGGS, AAY, and GPGPG) were used to construct the chimeric vaccine. CTL and B-cell epitopes were connected using the AAY and GPGPG linker, as reported by Robert et al. [65]. Besides acting as a proteasomal cleavage site, the AAY linker supports the epitope presentation by helping them find a suitable site for attachment on TAP transporters [66,67]. The GPGPG linker averts the formation of junctional immunity and promotes HTLs immune response, as demonstrated experimentally by Livingstone et al. [68] in mouse models. EAAAK linker was utilized to provide structural stiffness that can lessen the interference from other protein regions during the interaction between the adjuvant and its receptor [69]. In order to provide flexibility in the protein 3D structure, an alternative linker, GGGGS, was used [70]. Subunit vaccines are often less immunogenic and effective, requiring the inclusion of an adjuvant, which can promote and direct the adaptive immune response to the antigen (vaccine construct) [71]. Thus, we used 50S ribosomal protein L7/L12 to formulate a multi-epitope vaccine construct. Upon activation of naïve T-cells, the 50S ribosomal protein L7/L12 is reported to induce the maturation of DCs, CTLs, HTLs, and IFN-γ-producing cells [72].The refined modelled tertiary structure of the putative vaccine construct indicated a Z-score of −6.14, which corresponds to the X-crystallography-determined structures for proteins of similar sizes. Moreover, the predicted modelled vaccine structure in this study is comparable to the study of Droppa-Almeida et al. [73] and Rekik et al. [74], wherein the predicted tertiary structure of the constructed vaccines had a Z-score of −5.26 kcal/mol, and −9.51 kcal/mol, respectively. Therefore, the predicted 3D structure of the multi-epitope vaccine construct was deemed a high-quality one and was used for the downstream structure-based analysis.TLRs are Pattern Recognition Receptors (PRRs), which are expressed on both innate immune (DCs and macrophages) and non-immune cells (fibroblast cells and epithelial cells). They play an important role in innate immunity by identifying the conserved pathogen-associated molecular pattern (PAMP) derived from diverse pathogens [75]. TLR2 and TLR4 have also been studied to recognize the viral structural proteins, which results in the production of inflammatory cytokines against the viral infection [76,77,78]. TLR3 also detects viral infection and initiates an innate immune signalling pathway [79]. We carried out protein–protein docking to compute the binding affinity of the chimeric vaccine for TLRs. The docking analysis revealed the lowest (negative) S-scores (high-affinity) for the constructed vaccine–TLRs complexes. In addition, ionic and hydrogen bonding between the vaccine construct–TLRs interface also indirectly supported the formation of stable complexes [80], implying the potential of the designed vaccine construct to trigger an appropriate downstream immune pathway. Molecular dynamics simulation is a reliable approach for capturing motion at the atomic level, which is very difficult to accomplish by employing experimental methods [81]. The structural stability and compactness of the vaccine construct–TLRs complex were validated using the RMSD, RMSF, and Rg descriptors. The SASA profile of complexes indicated structural adjustments caused by the vaccine construct (ligand) binding to the receptor. Besides, H-bond analysis supported the formation of several long-lasting H-bonds between vaccine construct–TLRs interface that could play an essential role in complex stability [82]. Consistent with the docking analysis, binding free energy calculation with MM/GBSA method showed negative ∆G scores (energetically favourable binding) for peptides–HLA molecule complexes and modelled vaccine–TLRs complexes [83].The C-ImmSim server predicted high titers of neutralizing antibody production following in silico immunization, which is essential to combat the viral infection. Immune simulation findings also indicated substantial CTLs and IFN-γ levels, hence induction of long-lasting cellular and adaptive immune responses against CCHFV infection. These outcomes are comparable to other studies that designed multi-epitope vaccine constructs using B-cell, CTL, and HTL epitopes [32,63]. Nonetheless, experimental validation of the constructed vaccine is required to support the findings of the current investigation.

The present study represents an alternative approach to designing a vaccine based on a multi-epitope vaccine ensemble comprising antigenic components of CCHFV proteins (Asia-1 genotype) to tackle the antigenic complexity. Although numerous immunoinformatics-based methods were used, and the designed vaccine construct is predicted to be immunogenic; nevertheless, the extent of protection from the viral infection is unknown. Following the literature, the order and spacing of the CTL and B-cell epitopes were provided; still, this would require more proof to obtain the best possible immunogenicity with CTL and B-cell epitopes. The next step would be in vitro immunological assays to establish the immunogenicity of the designed vaccine construct and perform challenge-protection clinical experiments to validate the strategy.