Varicella zoster virus infects more than 90% of the worldwide population and is known to cause chickenpox, a childhood-acquired infection, and shingles, a latent infection [61]. Besides, the virus is also the second most causative agent for encephalitis [62, 63]. Currently available antiviral treatments haven’t shown any striking significance in preventing its infection [14, 15]. With the concept of attenuation in VZV being poorly understood, the existing vaccines aren’t full-fledged either. The virus present in attenuated vaccines gets activated, retaining the ability to infect neurons and thereby establishing latency [64–67]. Also considering the fact that these vaccines are not suitable for older adults, immunocompromised individuals, and pregnant women, there is a constant necessity to develop a high-efficacy epitope-based vaccine against VZV [68–71]. In addition to this, epitope-based vaccines are known for their better safety profiles and high logistic feasibility [72]. Since conventional development of a vaccine candidate requires laborious microbial, biochemical, and immunological steps as well as the necessity to handle viable pathogens, the immunoinformatic approach can be used for computationally determining the antigenic components to design such vaccines [73, 74]. This approach has been successful in designing vaccine candidates for several pathogens such as Epstein-Barr Virus [75], Human cytomegalovirus [76], Helicobacter pylori [77], Zika virus [78], HIV [79], and SARS-CoV2 [80].
Based on their ability to promote cell-cell fusion, trigger an immune response, and cause an infection, four envelope glycoproteins of the virus were considered for designing the vaccine [81–89]. Web-based servers were used for ‘epitope-fishing’, i.e., determining the epitopes from the selected proteins, typically the 9mer CTL epitopes and the 15mer HTL epitopes that are capable of activating Cytotoxic T cells and Helper T cells respectively [90]. Out of these, a total of 10 CTL epitopes and 10 HTL epitopes were screened and finalized for the construction of a multi-epitope vaccine, based on certain criteria such as - should be antigenic, should be non-allergic, should be immunogenic, should be highly promiscuous (binding towards ≥ 4 and ≥ 3 HLA molecules for CTL and HTL epitopes respectively), should have overlapping CTL and HTL epitopes and should have high affinity towards HLA alleles, as seen in their docking patterns [91–94]. A multi-epitope vaccine is preferred over the conventional single-epitope vaccines due to the following rationale: (a) Lesser adverse effects, since the unwanted antigenic components are eliminated; (b) Multiple T cells can be activated due to the presence of multiple HLA epitopes; (c) Ability to induce both cellular and humoral immunity due to the presence of overlapping CTL, HTL, and B cell epitopes; (d) Higher immunogenicity to the vaccine candidate due to the provision of having an adjuvant molecule linked with it [95–99]. The successful induction of T cell response by a synthetic epitope-based vaccine was first demonstrated by Aichele et al [100, 101]. Further works also inferred that the short peptides are capable of activating an early and efficient T-cell response; generally, the IFN-γ dominant Th1-type response [102, 103]. These Th1 cells in turn produce cytokines such as IFN-γ, IL-2, and TNF-α [101, 104, 105]. IFN-γ is a member of the type II interferon family that orchestrates numerous protective functions in elevating the immune response during viral infections [106, 107]. Moreover, the work done by Shakya et al. shows that IFN-γ inhibits the replication of VZV in a cell line-dependent manner [108]. We propose a mechanism for the action of the vaccine candidate in vivo to show its possible interactions and immune system activation strategy, as shown in Fig. 8.
In order to enhance the immunogenicity and the depot generation ability of this multi-epitope construct, there is the necessity to tag it with an adjuvant molecule. In the present study, CTB (Cholera toxin B) was used as an adjuvant, a toxin widely known for its inflammatory function, and extensively studied for having it conjugated with antigens [109]. Besides, CTB also acts as an activator of the TLR4 molecule, and their binding can be demonstrated using ELISA-based assays [42]. Linkers were employed for linking the multiple epitopes as well as the adjuvant into a single protein sequence. Taking into consideration the fact that they minimize junctional epitopes and enhance flexibility, as well as immunogenicity, the linkers GGGS and GPGPG, were used for linking the epitopes with each other [110–112]. In order to have the different domains separated, i.e., the adjuvant and the multi-epitope sequence, an EAAAK linker was used [113]. Also, the arrangement pattern of the epitopes was designed in such a way that no transmembrane helices shall arise from the construct, since they can eventually reduce the expression rate of the vaccine [114]. Physiochemical analyzes showed that the molecular weight of the 455 amino acids long final multi-epitope vaccine construct was 48279.67 Da. Theoretical pI for the vaccine construct was calculated to be 10.11. At 280nm in water, the extinction co-efficient of the vaccine molecule, i.e., a measure of how strongly its constituents absorb light, was found to be 38070 M− 1 cm− 1, assuming all pairs of Cys residues form cystines, and 37820 M− 1 cm− 1, assuming all Cys residues are reduced [115]. The half-life was estimated to be 30 hours, > 20 hours and > 10 hours for mammalian reticulocytes in vitro, yeast in vivo and E.coli in vivo respectively. The aliphatic index, which defines the relative volume occupied by aliphatic side chains, was found to be 79.34, indicating that the vaccine molecule is thermally stable [116, 117]. The instability index was computed to be 31.36, classifying the vaccine to be a stable one. The GRAVY (Grand average of hydropathicity) value of the vaccine was calculated to be -0.024. Since lower GRAVY values correspond to higher hydrophilicity, the vaccine was found to be highly soluble in water [118]. Besides, with an antigenic score of 0.5759, the vaccine was shown to be a potential antigen in nature and the allergenicity assessment inferred it to be a non-allergen. Post structural modeling of the vaccine, its validation was carried out using ProSA-web server, which predicted the Z-score of the model to be -7.23, a score lying within the range of native proteins of its comparable size, whose structures are experimentally determined using techniques such as X-ray crystallography and NMR [119].
TLRs are vital components in innate immunity; to date, many viruses are being shown to trigger an innate immune response via TLRs, suggesting their importance in viral infections [120]. Studies showed that the epitope-adjuvant complex used in the present study possesses efficient binding with TLR1 and TLR4; this was further confirmed by studies on BMDCs (Bone marrow-derived dendritic cells) by Hou et al [110, 121]. Hence, they were considered for further docking analyses. Herein, the chimeric form of TLR4 bound to an MD-2 was taken, because MD-2 is required for glycosylation that is essential for the cell surface expression of TLR4; besides, MD-2 also helps in enhancing the TLR4 mediated response towards the multi-epitope sequence [122–124]. Molecular analysis of the docking results showed that there were 2 salt bridges, 4 hydrogen bonds, and 183 non-bonded contacts between TLR1 and the vaccine construct. The hydrogen bonds were formed between Asp103, Ser34, Asp 61, and Leu42 of the TLR1 and His450, His445, Cys443, and Lys436 of the vaccine respectively. Likewise, 4 hydrogen bonds and 87 non-bonded contacts were shown between TLR4 and the vaccine construct. The hydrogen bonds were formed between Thr1300, Gln 162, Lys 21, and Ser 45 of the TLR4 and Gly429, His371, Leu444, and His447 of the vaccine respectively. On the other hand, the interaction pattern of MHC I and MHC IIwith the vaccine was also determined using molecular docking. It was seen that there were 5 salt bridges, 7 hydrogen bonds, and 96 non-bonded contacts between MHC I and the vaccine construct. The hydrogen bonds were formed between Glu353, Asp196, Glu128, Lys334 Asp335, and Thr228 of the MHC I and Asn231, Arg219, His448, His449, Tyr442, and His447 of the vaccine respectively. Likewise, 7 salt bridges, 6 hydrogen bonds and 88 non-bonded contacts were shown between MHC II and the vaccine construct. The hydrogen bonds were formed between Glu170, Asp169, Asn116, and Leu43 of the MHC II and Arg418, Arg419, Thr439, and His450 of the vaccine respectively.
The molecular dynamics simulation studies carried out for 100ns on the vaccine molecule showed that the vaccine was structurally stable and also flexible in terms of its RMSD and RMSF plots. The vaccine candidate sequence was then subjected to reverse translation in order to obtain the nucleotide sequence and analyze the expression efficiency after having it inserted into the pET-28a + expression vector, in the K12 strain of E.Coli bacteria. The 1365 nucleotide long optimized sequence showed a CAI (Codon adaptation Index) value of 0.918 and GC-Content of 53.7%. The results assured the multi-epitope vaccine to be efficiently expressed in bacteria, post cloning.