CHPV, which causes encephalitis, has been regarded as an emerging tropical pathogen with fatality rate of 55–77%, predominantly affecting children of age group 2–16 years [73]. There is no specific treatment available for the disease till date but a symptomatic treatment is done using mannitol to reduce the brain edema [16]. Hence, development of an effective vaccine has become the need of the hour. A recombinant vaccine has been developed using the complete G gene of the CHPV isolate [19]. A β-propio lactone (BPL) inactive tissue culture based vaccine has also been developed [74]. Unfortunately, for both types of vaccines developed so far, clinical trials have been performed only in mice but not in humans [10]. So far no vaccine is available against CHPV. Thus, aim of the present study was to design a multi-epitope, prophylactic vaccine targeting the CHPV glycoprotein which is primarily responsible for the virus envelopment, budding and antigenicity. Epitope-based vaccines represent a new strategy, developing immune response only against the selected epitopes, thus, avoiding the side effects of other unfavourable epitopes unlike the case of using complete antigen in recombinant vaccine technology [20, 75, 76].
The advances in the field of immunoinformatics, along with the knowledge of host immune response is being successfully used for the development of epitope based vaccines against various pathogens [77, 78]. Designing of epitope driven vaccine is a modern idea, which is successfully applied in several immunological studies for the development of vaccines [79]. Similarly, the present study is centred on designing of a multi-epitope vaccine because these vaccines have advantages over traditional and single-epitope vaccines due to the following unique features: i) TCRs from various T cell subsets can recognize the multiple MHC Class I and Class II epitopes ; ii) CTL, HTL and B cell epitopes may overlap, thus, it has the capacity to induce humoral and cellular immune responses simultaneously; iii) linking an adjuvant to the vaccine enhances the immunogenicity and provides long lasting immune response; iv) the likelihood of pathological immune responses or adverse effects is lowered because it is less likely to contain unwanted components [29,30,70,80–83)]. Further, it has been demonstrated that multi-epitope combined vaccine induce stronger CTL responses compared to those induced by a single-epitope vaccine by enhancing cellular immunity and releasing immune tolerance [29]. The cellular and humoral responses generated by the multi-epitope vaccines are highly specific with increased cytokines production [84–86]. A multi-epitope vaccine developed with such precautions can thus become a powerful tool in the battle against viral infections [87].
One of the troubles with the conventional approach of vaccine discovery is that many of the proteins expressed during infection are not always expressed in vitro, i.e., good candidate antigens might be overlooked [32]. In silico methods whereas, screens for all the probable candidate antigens, as predicted by various tools and algorithms which might otherwise remain undetected [32]. It is extremely important to pick the correct antigenic epitopes of the target proteins to be used in the building of multi-epitope vaccine through in silico methods [88]. The CTL, HTL and IFN-γ epitopes selected for the study were screened against various immunological filters (Table 1 & Table 2). The applied filters were: the epitopes should be promiscuous (bind with multiple MHC class I and MHC class II alleles), must be present on the surface of the target protein, must be immunogenic as well as antigenic. The promiscuous epitopes are those with sensitivity towards several HLA alleles. These epitopes are of paramount importance in vaccine formulation, as they are capable of developing an efficient immune response in the host, as they have affinity to several forms of HLA allele. Thus, the filtered out HLA class I and class II T cell epitopes were further evaluated for the study of promiscuity. In the present study, the epitopes expected to have a strong binding affinity to multiple HLAs were screened out and identified as promiscuous epitopes. The overlapping CTL and HTL epitopes have the potential to trigger cytotoxic T cells and helper T cells, which in turn generate immune responses. Allergenicity is one of the key issues faced during the production of vaccines. Hence, evaluation of allergenicity is necessary at an early stage of designing the vaccine. While developing the final vaccine model, the screened out epitopes were first subjected to an allergenicity assessment followed by the whole vaccine construct. The vaccine construct designed in this study was observed to be non-allergenic. Other physicochemical features like molecular weight, instability index, theoretical pI, aliphatic index, GRAVY score and half-life of the vaccine were also checked. The molecular weight of the vaccine was found to be 40 kDa and the instability index calculated was 13.11 which indicated that the designed vaccine is quite stable. Generally, a protein whose instability index is smaller than 40 is predicted to be stable and values above 40 predicts that the protein may be unstable [41]. Moreover, the vaccine has exhibited a fair percentage of solubility in over-expressed conditions. The GRAVY index of the vaccine was 0.083 indicating the vaccine’s polar nature and its effective interaction with water [89]. The high aliphatic index of 79.95 signified that the vaccine is a thermostable protein. The aliphatic index is commonly defined as the relative volume of a protein which is occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine) [90]. The half-life of the vaccine is 30 hours in mammalian reticulocytes, > 20 hours in yeast and > 10 hours in Escherichia coli. The half-life of a protein is defined as the time it takes for the concentration of the protein to be reduced by 50% after its synthesis in the cell. Similarly, Foroutan and his colleagues used the same array of in silico analysis in order to assess the allergenicity and physicochemical properties of their designed vaccine candidate against Toxoplasma gondii [91]. They have also performed laboratory validation of their vaccine candidate, which revealed that the multi-epitope vaccine was able to trigger strong humoral and cellular responses in mice [91]. The physicochemical properties predicted in our study were comparable to those predicted by Foroutan et al., in their recently published work [91]. In fact, the instability index and aliphatic index of our vaccine candidate was found to be better when compared to the values reported by [91]. The Ramachandran Plot, ERRAT score, Verify3D score and Z score analysis validated the overall quality of the vaccine construct (Fig. 5). Thus, after rigorous in silico analysis the final vaccine construct was designed. A similar approach was used by Bazhan and his co-workers, where they have designed a T-cell multi epitope vaccine against Ebola virus. The T-cell epitopes were predicted using IEDB – Immune Epitope Database and the vaccine candidate constructed using the suitable epitopes were found to be immunogenic when expressed in mice [92].
As the CHPV glycoprotein is an envelope protein, the Toll-like Receptor-4 (TLR4) and Toll-like Receptor-2 (TLR2) expressed in the plasma membrane of the cells should primarily recognize the structural components of the virus [93–95]. CHPV regulates TLR4, which leads to the secretion of pro-inflammatory cytokines and Nitric oxide (NO) in monocytes-macrophage cell line of mouse [96]. In humans, TLR4 is expressed in immune cells such as monocytes, macrophages, granulocytes and immature dendritic cells [97]. Cholera toxin B (CTB) has been proven to act as a potential viral adjuvant [98–100]. Activation of TLR4 by CTB is presumably due to the direct interaction between TLR4 and CTB [101]. This conclusion was supported by the evidence that the capacity of CTB to induce inflammatory response is lost in TLR4-deficient macrophages. CTB is able to induce NF-κB activation in TLR4 receptor cells by direct binding, which has been demonstrated using ELISA-based assays [101]. Further, TLR2 has been associated with the recognition of viral envelope glycoprotein [93]. The core TLR2 signalling pathway utilizes myeloid differentiation factor 88 (MyD88) as the primary adaptor, and results in NF-κB and mitogen-activated protein kinase (MAPK) activation as well as secretion of a core panel of cytokines [93]. It has also been reported that TLR2 acts as heterodimer with TLR1 for the generation of innate immune response and has been shown to recognise viral proteins [59, 93–95]. TLR1/TLR2 dimer generates intracellular signalling via IRAK4 mediated activation of IRAK1/2, which results in the activation of NF-κB, p38 and JNK proteins in the cytoplasm, involved in triggering innate immune response [102].
Figure13. Proposed mechanism of working (A) TLR signal transduction pathway: TLR1/TLR2 heterodimer or TLR 2/2 homodimer utilizes MyD88 and MAL as primary adapters to activate NF-κB that triggers inflammatory cytokine secretion. TLR4 uses four primary adapters namely MyD88, MAL, TRIF and TRAM for NF-κB secretion which in turn induce inflammatory cytokine secretion activating IFN pathway. (B) The CTB activates and interacts with TLR4, expressed on macrophages, B cells and monocytes which up regulate the cytokine secretion. The other immune cells, such as NK-cells, T cells or other human monocytes, will also indirectly be stimulated by CTB. Furthermore, the CTL and HTL epitopes interact with HLA class I and HLA class II and thus form epitope-HLA complexes which in turn interact with CTLs and HTLs, activate them and induce their proliferation. The IFN- γ will induce IFN genes. The proposed vaccine is thus capable of stimulating both adaptive and innate immunity.
Hence, the interaction pattern of multi-epitope vaccine against TLR4/TLR2/TLR1 was analyzed using Molecular Docking Analysis (Figs. 7, 8 & 9). The docking analysis of TLR4 and the vaccine construct showed that there is 1 salt bridge and 12 hydrogen bonds formed during this interaction. The docked complex showed that the salt bridge was formed between Glu53 of TLR4 and Arg303 of the vaccine. Out of the 12 hydrogen bonds, 10 bonds were formed between TLR4 and the vaccine, and remaining 2 bonds were formed between the MD-2 co-receptor protein and the vaccine. Thus, docking studies indicate that both TLR4 and MD-2 are responsible for a stable interaction of the vaccine. Molecular Dynamics Simulation (MDS) for both the vaccine and vaccine-TLR4 complex was performed in order to assess the stability of the vaccine and the complex at various thermo-baric conditions. MDS results indicated that both the structures remained stable at a temperature of 300K and a pressure of 1 bar. A trajectory analysis of 30 ns revealed that both the structures remained stable during the simulation run of 30 ns (Fig. 10). However, the RMSD value obtained for the vaccine-TLR4 complex was much lower when compared to the RMSD value for the vaccine, indicating stable interaction between the vaccine and TLR4 protein. The RMSF plot for the multi-epitope vaccine showed various regions of high flexibility for the vaccine, whereas the RMSF plot for the vaccine-TLR4 complex was almost stable with very little fluctuation. The highly fluctuating residues (Pro372, Leu383, Phe385, His387 and Thr388) obtained from the RMSF data of the vaccine showed very little fluctuation when compared to the RMSF data of the vaccine-TLR4 complex. This indicated that these residues tried to modify their interaction with the TLR4 and this modification led to the reinforcement of appropriate interactions between the vaccine and TLR4 protein, which led to the stability of the system. The plots for radius of gyration also showed little variation which indicated the compactness of the protein structures due to inter and intra molecular interactions between the vaccine and TLR4 protein. The efficient cloning and expression of such a multi-epitope vaccine in a suitable expression vector is again a very important step. Hence in the present study, an in silico cloning was performed to assure effective expression and translation of the designed multi-epitope vaccine construct into an expression vector: pET-28a (+) (Fig. 11). Several research groups have recently applied similar strategy to design a multi-epitope vaccine against Klebsiella pneumoniae [88], Kaposi Sarcoma [20], Pseudomonas aeruaginosa [103], Epstein Barr virus [104], Malaria [105], Hendra virus [106] and Nipah virus [107]. Similar approaches have also been used for developing vaccine against cancer antigens [22, 108–110] The proposed mechanism of action was also predicted for the final vaccine model (Fig. 13). Since, the vaccine comprised of CTL, HTL, and IFN-γ epitopes, it could trigger the stimulation of the respective immune cells in the host, which could further activate other immune cells by complex signaling.