Designing Novel Subunit Vaccines against Herpes Simplex Virus-1 using Reverse Vaccinology Approach

Herpes Simplex Virus (HSV) is an infectious virus that is responsible for various types of orofacial and genital infections. Two types of HSV viruses, HSV-1 and HSV-2, are the most dangerous HSV viruses. Every year, millions of people get infected with this menacing virus, however, no satisfactory treatment or vaccine has not yet been discovered to fight against HSV. Although there are some anti-viral therapies, however, studies have showed that such anti-viral therapies may also fail to provide good impact. In this study, a possible subunit vaccine against HSV-1, strain-17, was designed using the tools and reverse vaccinology and bioinformatics. Three potential antigenic envelope glycoproteins were selected from nine envelope glycoproteins, for possible vaccine construction. Potential epitopes capable of inducing high immunogenic response and at the same time have non-allergenicity and conservancy across other strains and species, were selected by some robust analysis, for vaccine construction. Finally, three possible vaccines were designed. Each of the vaccine construct differ from each other only in their adjuvant sequences and based on molecular docking analysis, one best vaccine construct was selected for molecular dynamics simulation study and in silico codon adaptation. The experiment showed that the selected best vaccine should be good candidate against HPV-1, strain-17. However, wet lab study should be conducted on the suggested vaccine(s) for confirming their potentiality, safety and efficacy.


Introduction
Herpes Simplex Virus (HSV) is a member of the herpesviridae family. HSV is a double stranded DNA containing virus with an icosapentahedral capsid. The HSV-1 and HSV-2 are the two most dangerous types of herpes. HSV-1 mainly causes the orofacial infections and HSV-2 usually causes genital infections [1,2]. The HSV-1 virus is transmitted from individual to individual primarily by oral-oral contact and cause a disease which is known as cold sores. HSV-1 infection is wide-spread throughout the world and in many cases, the infection may remain latent or undetected throughout the whole life of the infected person. Sometimes, the infection may lead to much dangerous diseases like the encephalitis. On the other hand, the HSV-2 is mainly a sexually transmitted virus and therefore, it mainly causes the genital herpes. According to a study, the total number of new HSV-1 cases in 2012 was estimated to be 118 million [3,4]. Moreover, the global incidence of HSV-2 was estimated to be about 417 million in 2012 [5]. Sometimes, the incidence of HSV infection leads to other diseases like the Alzheimer's disease and liver failure [6,7].
Currently, the treatment of HSV is carried out using many antiviral medications like acyclovir, valacyclovir, famciclovir etc. However, in many cases, such antiviral medications have failed to provide complete resistant to HSV and reduce the mortality rate of the HSV infected patients.
Moreover, studies have proved that the viral resistance of Herpes virus to such medications (like the acyclovir) may also occur and such incidence can make the treatments more complicated [8,9,10,11]. Although researchers are working on to develop an effective vaccine against the Herpes virus like subunit vaccines, inactivated virion vaccines, genetically attenuated vaccines etc., to date, no satisfactory vaccine has been successfully entered the market [12]. In this study, the reverse vaccinology and bioinformatics approach was used to develop an effective Herpes vaccine against the HSV-1, strain-17 (Figure 01). Reverse vaccinology is defined as the process of vaccine development where the novel antigens of a virus or organism are identified by analyzing the genomic information of that particular organism or a virus. In reverse vaccinology, various tools of bioinformatics are used for identifying the novel antigens useful for vaccine development by dissecting the genome as well as for studying the genetic makeup of a pathogen. This approach of vaccine development also helps the scientists to understand which antigenic segments of a virus or pathogen should be given emphasis during the vaccine development. This method is a quick easy and cost-effective way to design vaccine. Reverse vaccinology is successfully used for developing vaccines for many viruses like the Zika virus, Chikungunya virus etc. [13,14].

Materials and methods:
The experiment focuses on developing of vaccines against the Human Herpes Simplex Virus-1 (strain-17).

Strain Identification and Selection
The Herpes Simplex Virus-1 (strain-17) was identified and selected by analyzing and reviewing different entries of the online server of National Center for Biotechnology Information or NCBI (https://www.ncbi.nlm.nih.gov/).

Antigenicity Prediction and Physicochemical Property Analysis of the Protein Sequences
The antigenicity of the protein sequences were predicted by the online server, VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.htm), keeping the threshold at 0.4 and tumor model was used [15,16,17]. Only the antigenic proteins were selected for further analysis.

T-cell and B-cell Epitope Prediction
The T-cell and B-cell epitopes of the selected protein sequences were predicted using online epitope prediction server Immune Epitope Database or IEDB (https://www.iedb.org/). The IEDB database contains huge amount of experimental data on T-cell epitopes as well as antibodies. These data are collected from various studies that are conducted on human, non-human primates and other animals. It is a server that allows robust analysis on various epitopes by exploiting various tools: population coverage, conservation across antigens and clusters with similar sequences [19].
The MHC class-I restricted CD8+ cytotoxic T-lymphocyte (CTL) epitopes of the selected sequences were obtained using NetMHCpan EL 4.0 prediction method for HLA-A*11-01 allele.
The MHC class-II restricted CD4+ helper T-lymphocyte (HTL) epitopes were obtained for HLA DRB1*04-01 allele using Sturniolo prediction method. The top ten MHC class-I were selected based on their percentile scores and antigenicity scores (AS). Moreover, MHC class-II epitope prediction generated several epitopes with similar AS and percentile scores. However, 2 epitopes were taken from each of the top five categories. On the other hand, five random B-cell lymphocyte epitopes (BCL) were selected based on their length (the epitope sequences that had ten amino acids or above were selected) and obtained using Bipipered linear epitope prediction method, keeping the parameters default.

Transmembrane Topology and Antigenicity Prediction of the Selected Epitopes
The transmembrane topology of the selected epitopes were determined using the transmembrane topology of protein helices determinant, TMHMM v2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). The server predicts whether the epitope would be transmembrane or it would remain inside or outside of the membrane [20]. The antigenicity of the selected epitopes were predicted using VaxiJen v2.0 (http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.htm), using the tumor model and threshold of 0.4.

Conservancy Prediction of the Selected Epitopes
The conservancy analysis of the selected epitopes were performed using the 'epitope conservancy analysis' tool of IEDB server (https://www.iedb.org/conservancy/) [19]. The sequence identity threshold was kept '>=50'. For the conservancy analysis of the selected epitopes of the Herpes across the species as well as strains. Based on the antigenicity, allergenicity, toxicity and conservancy analysis, the best ligands were selected for the further analysis and vaccine construction. The epitopes that showed antigenicity, non-allergenicity, non-toxicity and high (more than 90%) conservancy and more than 50% minimum identity, were considered as the best epitopes. For B-cell epitope selection, only the antigenic and non-allergenic epitopes were taken for further analysis.

Cluster Analysis of the MHC Alleles
Cluster analysis of the MHC alleles helps to identify the alleles of the MHC class-I and class-II molecules that have similar binding specificities. The cluster analysis of the MHC alleles were carried out by online tool MHCcluster 2.0 (http://www.cbs.dtu.dk/services/MHCcluster/) [23].
During the analysis, the number of peptides to be included was kept 50,000, the number of bootstrap calculations were kept 100 and all the HLA supertype representatives (MHC class-I) and HLA-DR representatives (MHC class-II) were selected. For analyzing the MHC class-I alleles, the NetMHCpan-2.8 prediction method was used. The output of the server generated results in the form of MHC specificity tree and MHC specificity heat-map.

Molecular Docking of the Selected Epitopes
The GGGS linkers were used to attach the PADRE sequence with the CTL epitopes and the CTL epitopes with the other CTL epitopes, GPGPG linkers were used to connect the CTL with HTL epitopes and also the HTL epitopes among themselves. The KK linkers were used for conjugating the HTL and BCL epitopes as well as the BLC epitopes among themselves [32,33,34,35,36,37,38]. Studies have proved that the PADRE sequence improves the CTL response of the vaccines that contain it [39]. Total three vaccines were constructed in the experiment.

Antigenicity, Allergenicity and Physicochemical Property Analysis
The antigenicity of the constructed vaccines were determined by the online server VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.htm). The threshold of the prediction was kept at 0.4. AlgPred (http://crdd.osdd.net/raghava/algpred/) and AllerTop v2.0 (https://www.ddg-pharmfac.net/AllerTOP/) were used for the prediction of the allergenicity of the vaccine constructs. The AlgPred server predicts the possible allergens based on similarity of known epitope of any of the known region of the protein [40]. MEME/MAST motif prediction approach is used in the allergenicity prediction of the vaccines by AlgPred. Moreover, various physicochemical properties of the vaccines were examined by the online server ProtParam (https://web.expasy.org/protparam/).

Secondary and Tertiary Structure Prediction of the Vaccine Constructs
The secondary structures of the vaccine constructs were generated using online tool PRISPRED (http://bioinf.cs.ucl.ac.uk/psipred/). PRISPRED is a simple secondary structure generator which can be used to predict the transmembrane topology, transmembrane helix, fold and domain recognition etc. along with the secondary structure prediction [41,42]. The PRISPRED 4.0 prediction method was used to predict the secondary structures of the vaccine constructs. The βsheet structure of the vaccines were determined by another online tool, NetTurnP v1.0 (http://www.cbs.dtu.dk/services/NetTurnP/) [43]. The tertiary or 3D structures of the vaccines were generated using online tool RaptorX (http://raptorx.uchicago.edu/) server. The server is a fully annotated tool for the prediction of protein structure, the property and contact prediction, sequence alignment etc. [44,45,46].

3D Structure Refinement and Validation
The 3D structures of the constructed vaccines were refined using online refinement tool, 3Drefine (http://sysbio.rnet.missouri.edu/3Drefine/). The server is a quick, easy and efficient tool for protein structure refinement [47]. For each of the vaccine, the refined model 1 was downloaded for validation. The refined vaccine proteins were then validated by analyzing the Ramachandran plots which were generated using the online tool, PROCHECK (https://servicesn.mbi.ucla.edu/PROCHECK/) [48,49].

Vaccine Protein Disulfide Engineering
The vaccine protein disulfide engineering was carried out by online tool Disulfide by Design 2 v12.2 (http://cptweb.cpt.wayne.edu/DbD2/). The server predicts the possible sites within a protein structure which have the greater possibility of undergoing disulfide bond formation [50]. When engineering the disulfide bonds, the intra-chain, inter-chain and Cβ for glycine residue were selected. The χ3 Angle was kept -87° or +97° ± 5 and Cα-Cβ-Sγ Angle was kept 114.6° ±10.

Protein-Protein Docking
In

Molecular Dynamic Simulation
The molecular dynamics simulation study was performed on the best selected vaccine, HV-1. The study was carried out by the online server iMODS (http://imods.chaconlab.org/). iMODS can be used efficiently to investigate the structural dynamics of the protein complexes, since it is a quick, easy and user-friendly server. The server provides the values of deformability, B-factor (mobility profiles), eigenvalues, variance, co-variance map and elastic network, for a protein or docked protein-protein complex. The deformability depends on the ability to deform at each of its amino acid. The eigenvalue is related with the energy that is required to deform the given structure and the lower eigenvalue corresponds to the easier the deformability of the complex. The eigenvalue also represents the motion stiffness of the protein complex. The server is a fast and easy tool for determining and measuring the protein flexibility [66,67,68,69,70]. For analysing the molecular dynamics simulation of the HV-1-TLR-3 docked complex was used. The docked PDB files were uploaded to the iMODS server and the results were displayed keeping all the parameters as default.

Codon Adaptation and In Silico Cloning
The codon adaptation and in silico cloning were carried out for the best selected vaccine protein, HV-1. For conducting these experiments, the vaccine protein was reverse transcribed to the possible DNA sequences. The DNA sequence should encode the target vaccine protein. Later, the reverse transcribed DNA sequences were adapted according to the desired organism, so that the cellular mechanisms of that specific organism could use the codons of the adapted DNA sequences efficiently and provide better production of the desired product. Codon adaptation is a necessary step of in silico cloning since the same amino acid can be encoded by different codons in different organisms, a phenomenon which is known as codon biasness. Moreover, the cellular mechanisms of an organism may be different from another organism and a codon for a specific amino acid may not work in another organism. For this reason, codon adaptation step is performed that can predict the suitable codon that can encode a specific amino acid in a specific organism [71,72]. The codon adaptation of the selected vaccine protein was carried out by the Java Codon Adaptation Tool or JCat server (http://www.jcat.de/) [71]. Eukaryotic E. coli strain K12 was selected and rhoindependent transcription terminators, prokaryotic ribosome binding sites and SgrA1 and SphI cleavage sites of restriction enzymes, were avoided. In the JCat server, the protein sequences were reverse translated to the optimized possible DNA sequences. The optimized DNA sequences were taken and SgrA1 and SphI restriction sites were conjugated at the N-terminal and C-terminal sites, respectively. Next, the SnapGene [73] restriction cloning module was used to insert the new adapted DNA sequences between SgrA1 and SphI of pET-19b vector.

Identification, Selection and Retrieval of Viral Protein Sequences
The Herpes Simplex Virus, strain-17 was identified and selected from the NCBI

Antigenicity Prediction and Physicochemical Property Analysis of the Protein Sequences
From the selected nine proteins, only the antigenic proteins were selected for vaccine construction.
VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.htm) server was used for antigenicity determination. Among the nine protein, envelope glycoprotein E, envelope glycoprotein B and envelope glycoprotein D were determined as possible antigenic proteins by the server ( Table 01). The physicochemical property analysis was conducted on these three proteins.
The physicochemical study revealed that envelope glycoprotein B had the highest extinction coefficient of 105255 M -1 cm -1 and lowest GRAVY value of -0.403. However, all the three proteins showed instability and all of them had half-lives of 30 hours in the mammalian cell culture system.
The results of the physicochemical property analysis are listed in Table 02.

T-cell and B-cell Epitope Prediction and Topology Determination of the Epitopes
The T-cell epitopes of MHC class-I of the three proteins were determined by NetMHCpan EL 4.0 prediction method of the IEDB (https://www.iedb.org/) server keeping the sequence length 09.
The server yielded over 100 such epitopes. However, based on analyzing the best antigenicity scores (AS) and percentile scores for each epitope, the top ten potential epitopes were selected randomly for antigenicity, allergenicity, toxicity and conservancy tests. The server ranks the predicted epitopes based on the ascending order of percentile scores. The lower the percentile, the better the binding affinity. The T-cell epitopes of MHC class-II (HLA DRB1*04-01 allele) of the proteins were also determined by IEDB (https://www.iedb.org/) server, where the Sturniolo prediction method was used. Among the hundreds of epitopes generated by the server, best ten epitopes were selected for further analysis. Moreover, the B-cell epitopes of the proteins were selected using Bipipered linear epitope prediction method of the IEDB (https://www.iedb.org/) server and epitopes were selected based on their higher lengths (Figure 02). The topology of the selected epitopes were determined by TMHMM v2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Table 03 and Table 04 list the potential T-cell epitopes of envelope protein E, Table 05 and Table 06 list the potential T-cell epitopes of envelope glycoprotein B, Table 07 and Table 08 list the potential T-cell epitopes of envelope protein D and Table 09 list the potential B-cell epitopes with their respective topologies.

Antigenicity, Allergenicity, Toxicity and Conservancy Analysis
In the antigenicity, allergenicity, toxicity and conservancy analysis, the T-cell epitopes that were found to be highly antigenic as well as non-allergenic, non-toxic, had minimum identity of over 50% and had conservancy of over 90% and the antigenic as well as non-allergenic B-cell epitopes were selected for further analysis and vaccine construction. Among the ten selected MHC class-I epitopes and ten selected MHC class-II epitopes of envelope glycoprotein E, total six epitopes (three epitopes from each of the category) were selected based on the mentioned criteria: YTLSVGDIK, ITISTAAQY, AVKSRASGK, VRMETPEAILFS, LRFDVPTSCAEM and PSWVSLMPPKQV. On the other hand, among the ten selected MHC class-I epitopes and ten selected MHC class-II epitopes of envelope glycoprotein B, total six epitopes (three epitopes from each of the category) were selected based on the mentioned criteria: KVTDMVMRK, YAYSHQLSR, ASANASVER, TMYYKDVTVSQV, YARDLTTKARAT and VMRLQSNPMKAL. Moreover, like these proteins, six epitopes that obeyed the mentioned criteria, were selected for further analysis from the envelope glycoprotein D epitopes: KMADPNRFR, KIAGWHGPK, SIQDAATPY, QAYQQGVTVDSI, YWMRRHTQKAPK and VLLNAPSEAPQI. For the selection of the B-cell epitopes, the highly antigenic and nonallergenic sequences were taken for vaccine construction. Three epitopes from each of the protein category were selected. For this reason, total nine epitopes B-cell epitopes were selected for vaccine construction, since they obeyed the selection criteria. (Table 03, Table 04, Table 05,   Table 06, Table 07, Table 08 and Table 09).

Cluster Analysis of the MHC Alleles
The cluster analysis of the possible MHC class-I and MHC class-II alleles that may interact with the predicted epitopes of the three selected proteins, were performed by online tool MHCcluster 2.0 (http://www.cbs.dtu.dk/services/MHCcluster/). The tool generates the relationship of the clusters of the alleles in phylogenetic manner. Figure 03 illustrates the outcome of the experiment where the red zone indicates strong interaction and the yellow zone corresponds to weaker interaction.

Generation of the 3D Structures of the Epitopes and Peptide-Protein Docking
The 3D structures of the selected T-cell epitopes were generated by the PEP-FOLD3 server. The

Constructs
The three vaccines were found to be potent antigen as well as non-allergen. Since they are found to be non-allergenic, they are safe to use. In the physicochemical property analysis, the number of amino acids, molecular weight, extinction coefficient (in M-1 cm-1), theoretical pI, half-life, aliphatic index and GRAVY were determined. All the vaccines quite similar theoretical pI and ext.  Table 12.

Secondary and Tertiary Structure Prediction of the Vaccine Constructs
The secondary structures of the three vaccine constructs were generated by the online tools, PRISPRED (http://bioinf.cs.ucl.ac.uk/psipred/) and NetTurnP v1.0 (http://www.cbs.dtu.dk/services/NetTurnP/). From the secondary structure analysis, it was analyzed that, the HV-1 had the highest percentage of the amino acids (74.1%) in the coil formation as well as the highest percentage of amino acids (10.8%) in the beta-strand formation. However, HV-3 had the highest percentage of 30.1% of amino acids in the alpha-helix formation (Figure 05 and Table 13).
The 3D structures of the vaccine constructs were predicted by the online server RaptorX (http://raptorx.uchicago.edu/). All the three vaccines had 3 domains and HV-2 had the lowest pvalue of 8.91e-05. The homology modeling of the three dengue vaccine constructs were carried out using 1KJ6A (for HV-1), 1DD3A (for HV-2) and 4TQLA (for HV-3) as templates from protein data bank (https://www.rcsb.org/). The results of the 3D structure analysis are listed in Table 14 and illustrated in Figure 06.  Table 14. Results of the tertiary structure analysis of the vaccine constructs.

Protein Disulfide Engineering
In protein disulfide engineering, disulfide bonds were generated for the 3D structures of the vaccine constructs. The DbD2 server identifies the pairs of amino acids that have the capability to form disulfide bonds based on the given selection criteria. In this experiment, we selected only those amino acid pairs that had bond energy value was less than 2.00 kcal/mol. The HV-1 generated 12 amino acid pairs that had the capability to form disulfide bonds. However, only 4 pairs were selected since they had bond energy, less than 2.00 kcal/mol: 19 Arg and 148 Gly, 21 Gly

Protein-Protein Docking Study
The protein-protein docking study was carried out to find out the best constructed Herpes vaccine.
From analyzing the protein-protein docking, it was declared that HV-1 was the best constructed vaccine with the best and lowest scores in the MM-GBSA study and HawkDock study.  Table   15). The molecular dynamics simulation study and in silico codon adaptation studies were conducted on only the HV-1 vaccine.

Codon Adaptation and In Silico Cloning
For in silico cloning and plasmid construction, the protein sequences of the best selected vaccines were adapted by the JCat server (http://www.jcat.de/).

Discussion
Vaccine is one of the most important and widely produced pharmaceutical products. Millions of people and infants are getting vaccinated every year. However, the research and development processes of vaccines are costly and sometimes, it takes many years to develop a proper vaccine candidate against a particular pathogen. In recent times, various methods and tools of bioinformatics, immunoinformatics and reverse vaccinology are exploited for vaccine development, which save time and cost of the vaccine development process [76].
The current study was conducted to design and construct potential Herpes Simplex Virus (HSV), spread the virus from cell to cell as well as the fusion of the cells [81,82,83]. The envelope glycoprotein D also helps the virus to enter into the host cell [84]. The envelope glycoprotein C aids in the adsorption of the virion to the host cell surface [85]. Since, the glycoproteins mainly function in the infection process of the HSV, for this reason, targeting these glycoproteins could be potential strategy for vaccine construction.
After selecting the proteins, the antigenicity of the proteins were determined by VaxiJen V2.0 server. The proteins that were found to be antigenic, were selected for further analysis. Three glycoproteins, envelope glycoprotein E, envelope glycoprotein B and envelope glycoprotein D were found to be antigenic and for this reason, they were selected for further analysis. The various physicochemical properties like number of amino acids, molecular weight, theoretical pI, extinction co-efficient, instability index, aliphatic index, GRAVY were determined by ProtParam server. The IEDB server generates and ranks the T cell epitopes based on their antigenicity scores (AS) and percentile scores. The top ten MHC class-I epitopes were taken for analysis. However, since more than one epitope showed similar AS and percentile score, two epitopes were selected from each of the AS and percentile score categories. However, based on analyzing the lengths of the B-cell epitopes, five epitopes were selected for further analysis. The transmembrane topology of the selected eiptopes were predicted to determine whether they would be present at the exterior or interior of the viral envelope.
Antigenicity is defined as the ability of a foreign substance to act as antigen and activate the T cell and B cell responses, through their antigenic determinant portion or epitope [91]. The allergenicity of a substance is defined as the ability of that substance to act as allergen and induce potential allergic reactions within the body [92]. On the other hand, when designing a vaccine, epitopes of that remain conserved across various strains, are given much priority than genomic regions that are highly variable among the strains since the conserved epitopes of protein(s) provide broader protection across various strains and sometimes even species [93]. When selecting the best epitopes for vaccine construction, some criteria were maintained: the epitopes should be highly antigenic, so that they can induce high antigenic response, the epitopes should be non-allergenic in nature, for this reason, they would not be able to induce any allergenic reaction in an individual and the epitopes should be non-toxic. The epitopes with 100% conservancy and over 50% minimum identity were selected for vaccine construction, so that, the conserved epitopes would be able to provide protection against various strains. The conservancy analysis of the epitopes were carried out using Human Herpes Simplex Virus-1 strain-F and Human Herpes Simplex Virus-2 strain-HG52, for comparison, so that the conservancy analysis across the strains as well as species could be carried out efficiently. Total 18 T-cell epitopes (six epitopes from each of the proteins) and 9 B-cell epitopes were selected for vaccine construction. Moreover, the cluster analysis of the MHC class-I alleles and MHC class-II alleles were also carried out to determine their relationship with each other and cluster them functionally based on their predicted binding specificity [94].
In the next step, the protein-peptide docking was carried out between the selected epitopes and the MHC alleles . The MHC class-I epitopes were docked with the MHC class-I molecule (PDB ID: 5WJL) and the MHC class-II epitopes were docked with the MHC class-II molecule (PDB ID: 5JLZ). The protein-peptide docking was performed to determine the ability of the epitopes to bind with their respective MHC molecule. After 3D structure generation of the epitopes, the docking was carried out by PatchDock and FireDock servers. YTLSVGDIK, ASANASVER, KIAGWHGPK, PSWVSLMPPKQV, TMYYKDVTVSQV and QAYQQGVTVDSI generated the best scores in the protein-peptide docking. However, all of the selected T-cell epitopes showed significant interaction capability with their respective targets.
After successful protein-peptide docking, the vaccine construction procedure was carried out.
Three different vaccines were constructed, that differ from each other based on their adjuvant sequences. The three vaccine constructs were designated as: HV-1 (600 amino acids long), HV-2 (685 amino acids long) and HV-3 (714 amino acids long).
After the vaccine construction was performed, the antigenicity, allergenicity and physicochemical property analysis were carried out. All the vaccine constructs were proved to be antigenic, as well as non-allergenic, for this reason, they should not cause any allergenic reaction within the body, however, all of them should be able to induce high immunogenic response. The extinction coefficient corresponds to the amount of light, that is absorbed by a compound at a certain wavelength [95,96]. HV-3 had the highest extinction co-efficient of 74566.83 M-1 cm-1. The aliphatic index of a protein referes to the relative volume occupied by the aliphatic amino acids in the side chains like alanine, valine etc. [97]. HV-2 had the highest aliphatic index among the vaccine constructs, although HV-3 had aliphatic index that is very near to the aliphatic index of HV-2. All the three vaccine constructs had the similar half-life of 1 hour in the mammalian cell culture and all of them were stable. All of the constructs had quite similar theoretical pI. Quite similar performances were observed by the three vaccine constructs in the physicochemical property analysis.
Two online servers, PRISPRED (http://bioinf.cs.ucl.ac.uk/psipred/) and NetTurnP v1.0 (http://www.cbs.dtu.dk/services/NetTurnP/), were used for protein secondary structure determination. HV-3 had the highest percentage of amino acids in the alpha formation and lowest percentage of amino acids in the beta-sheet formation as well as in coil structure formation. The 3D structures were generated by the server RaptorX (http://raptorx.uchicago.edu/) server and with the lowest p-value of 8.91e-05, HV-2 showed the best performance on the protein 3D structure generation. However, all of the proteins had 3 domains. After 3D structure generation, the 3D structures were refined by online tool 3Drefine (http://sysbio.rnet.missouri.edu/3Drefine/) and validated by PROCHECK (https://servicesn.mbi.ucla.edu/PROCHECK/) server. HV-3 had the best performance in the 3D structure refinement and validation study with 74.7% of the amino acids in the most favored regions and 23.7% of the amino acids in the additional allowed regions.
After validation of the 3D protein structures, the disulfide engineering of the vaccine constructs were performed using Disulfide by Design 2 v12.2 (http://cptweb.cpt.wayne.edu/DbD2/) server and the amino acid pairs with binding energy value less than 2.0 kcal/mol, were chosen for disulfide bond formation. With six pairs of amino acids, that had binding energy less than 2.0 kcal/mol, it can be considered that, HV-2 showed the best performance in the protein disulfide engineering study.
In the next step, the protein-protein docking was carried out using the vaccine constructs as ligands and various MHC alleles as receptors. The docking experiment was carried out to determine the best vaccine construct among the three vaccines. All the vaccines successfully bound with their target receptors. However, from the docking experiment, it was revealed that, HV-1 was the best vaccine among the three vaccine constructs. For this reason, HV-1 vaccine construct was used for molecular dynamics simulation and in silico cloning. The molecular dynamics simulation study showed that, the HV-1-TLR-3 complex had very low chance of deformability. This was further confirmed by the deformability graph of 10b where the spikes in the graphs rarely reaches to the maximum value. The eigenvalue of the complex of 1.042621e-04 also pointed to the fact that the HV-1-TLR-3 docked complex should be quite stable and should have relatively less chance of deformability. The Figure 10f illustrates the correlated motion of the amino acid residue pairs in red color and according to the figure, the complex showed a large amount of amino acid pairs in the correlated motion. Finally the HV-1 vaccine was reverse transcribed into the possible DNA sequence for cloning into the E. coli strain K12. Before in silico cloning procedure was performed, codon adaptation of the HV-1 vaccine was carried out, where the CAI value of 0.973 was achieved, which reflects that the newly adapted sequence had high degree of codons that are mostly likely to be used by E. coli strain K12. Moreover, good GC content of 56.33% was also achieved. Finally, the adapted DNA sequence was inserted into the pET-19b plasmid and the plasmid with the HV-1 insert was now 7372 base pairs long. This newly generated plasmid with the target DNA sequence can be inserted into the E. coli strain K12 for efficient production of the HV-1 vaccine.
However, more in vitro and in vivo researches should done to finally confirm the safety, efficacy and potentiality of the predicted vaccines.

Conclusion
Herpes Simplex Virus is one of the most infectious and sexually transmitted diseases in the world.
However, only the HSV-2 is transmitted sexually. The other type of Herpes virus, HSV-1, transmits though oral-oral contact. With very high infection rate, HSV can be considered as one of the fatal viruses in the world. However, no vaccines are yet discovered with satisfactory results, to control the infection of HSV. In this study, a possible subunit vaccine to fight against HSV-1, strain-17, was designed using various tools of bioinformatics, immunoinformatics and vaccinomics. In this study, first the potential proteins of the viral structure were first identified.
Next, the potential epitopes were identified through robust processes and these epitopes were used for vaccine construction. Since only the highly antigenic and at the same time, non-allergenic epitopes were selected, the constructed vaccines should confer very strong antigenic response, however, with no allergenicity, the constructed vaccines should be safe to be administered. Three possible vaccines were constructed and by conducting the docking experiments, one best vaccine construct was identified. Later, the molecular dynamics simulation study and codon adaptation as well as in silico cloning were performed for large scale production of the vaccines. The vaccine should provide better immunity towards the HSV-1, strain-17 as well as the HSV-1 strain-F and the HSV-2, strain-HG52, due to its conservancy across the strains and species. However, wet lab researches should be carried out on the findings of this experiment to finally confirm the safety, efficacy and potentiality of the vaccine constructs. Hopefully, this research will raise interests among the scientists of the respective field.