Computer-Aided Multi-Epitope Based Vaccine Design Against Monkeypox Virus Surface Protein A30L: An Immunoinformatics Approach

Monkeypox, a viral zoonotic disease resembling smallpox, has emerged as a significant national epidemic primarily in Africa. Nevertheless, the recent global dissemination of this pathogen has engendered apprehension regarding its capacity to metamorphose into a sweeping pandemic. To effectively combat this menace, a multi-epitope vaccine has been meticulously engineered with the specific aim of targeting the cell envelope protein of Monkeypox virus (MPXV), thereby stimulating a potent immunological response while mitigating untoward effects. This new vaccine uses T-cell and B-cell epitopes from a highly antigenic, non-allergenic, non-toxic, conserved, and non-homologous A30L protein to provide protection against the virus. In order to ascertain the vaccine design with the utmost efficacy, protein–protein docking methodologies were employed to anticipate the intricate interactions with Toll-like receptors (TLR) 2, 3, 4, 6, and 8. This meticulous approach led the researchers to discern an optimal vaccine architecture, bolstered by affirmative prognostications derived from both molecular dynamics (MD) simulations and immune simulations. The current research findings indicate that the peptides ATHAAFEYSK, FFIVVATAAV, and MNSLSIFFV exhibited antigenic properties and were determined to be non-allergenic and non-toxic. Through the utilization of codon optimization and in-silico cloning techniques, our investigation revealed that the prospective vaccine exhibited a remarkable expression level within Escherichia coli. Moreover, upon conducting immune simulations, we observed the induction of a robust immune response characterized by elevated levels of both B-cell and T-cell mediated immunity. Moreover, as the initial prediction with in-silico techniques has yielded promising results these epitope-based vaccines can be recommended to in vitro and in silico studies to validate their immunogenic properties.


Introduction
The global outbreak of monkeypox in 2022, encompassing both endemic and non-endemic regions, has ignited significant worldwide attention.Monkeypox virus (MPXV), a double-stranded DNA virus closely related to Vaccinia and cowpox viruses, has emerged as the causative agent.The virus exhibits a characteristic dumbbell-shaped core, measuring between 200 and 250 nm in size.It belongs to the Poxviridae family, specifically the Chordopoxvirinae subfamily, and is classified within the genus "Orthopoxvirus."Notably, the Orthopoxvirus genus comprises other notable members including Vaccinia virus, Cowpox virus, Camelpox virus, Rabbitpox virus, Horsepox virus, Ectromelia virus, and Variola virus [1].
In 1959, during the transportation of cynomolgus monkeys from Singapore to Denmark, a previously neglected pathogenic virus was discovered.Subsequently, various species including squirrels, rodents, mice, monkeys, dogs, and humans have all succumbed to infections caused by this virus [2].However, the inaugural case of monkeypox in the annals of human history was reported in 1970 within the Basankusu Territory of the Democratic Republic of the Congo, situated in Central Africa.Remarkably, this instance involved an unvaccinated newborn merely nine months old [3].It is worth noting that two distinct genetically divergent clades have been identified: the West African clade and the Central African clade, predominantly present in the Congo basin [4].
Over the past fifty years, isolated cases of monkeypox have predominantly emerged in African nations.To date, thousands of individual cases have been meticulously documented.Due to factors such as tourism and the transportation of livestock carrying the virus, sporadic occurrences and localized outbreaks have been reported in regions where the virus is not typically endemic [5].Theoretically, the monkeypox virus, along with other zoonotic poxviruses, could gradually assume the ecological niche once occupied by the closely related variola virus.Historically, the mortality rate associated with monkeypox infections has ranged from 1 to 10%, with children representing the majority of fatalities [6].Although the symptoms of monkeypox bear similarities to those of smallpox, the transmissibility and severity of the disease are comparatively milder.Since the eradication of smallpox in 1980 and the discontinuation of smallpox vaccinations, the monkeypox virus has emerged as the most significant orthopoxvirus responsible for persistent infections across various host species [6].
Nations in West and Central Africa, particularly those in close proximity to tropical rainforests, have occasionally reported outbreaks of monkeypox.Notably, the first instance of monkeypox outside of Africa was identified in the United States in 2003, signifying a noteworthy geographical expansion.While traditionally prevalent in Central and Western Africa, recent occurrences of human-to-human transmission and increased disease transmissibility have underscored the potential global ramifications of monkeypox, further complicating the global response to the COVID pandemic [7].Considering monkeypox's rapid proliferation and its presence in more than 70 locations, it seems like the infection has already been spreading at elevated levels, which have eluded detection by monitoring systems until now [8].
The viral pathogenicity of monkeypox entails an initial entry into the lymph nodes, followed by dissemination to various organs, resulting in a broad spectrum of symptoms.These symptoms encompass lymphadenopathy, influenzalike manifestations, and the formation of lesions that initially appear in the oropharynx and subsequently progress to involve other cutaneous regions, including the palms and soles [1].The onset of monkeypox-associated symptoms typically manifests within a three-week timeframe subsequent to viral infection.Following the onset of flulike prodromal symptoms, individuals typically develop a rash within one to four days, which initially resembles acne or vesicles.This rash persists for a duration of two to four weeks, progressing through several stages including the formation of scabs.The rash may manifest on or in close proximity to the genitalia, as well as other areas such as the feet, chest, and face.The contagiousness of the disease begins upon the appearance of initial symptoms and persists until the complete resolution of the rash, the shedding of all scabs, and the regeneration of a new layer of skin.Complications associated with monkeypox include secondary microbial infections, hypo-or hyperpigmentation resulting in enduring scars on the skin, corneal scarring, pneumonia, sepsis, and potential mortality [9].
Multiple observational studies have demonstrated that administration of the smallpox vaccine confers approximately 85% protection against monkeypox.Notably, in 2019, a vaccine targeting monkeypox, based on a modified attenuated vaccinia virus known as the Ankara strain, received approval.This immunization necessitates two separate doses; however, its accessibility remains limited.Vaccines against both smallpox and monkeypox are typically formulated using compositions derived from the vaccinia virus, owing to the cross-protection offered by orthopoxviruses [10].Within monkeypox viruses, the A30L protein serves as an envelope protein that plays a pivotal role in viral entry into host cells and facilitates cell-cell fusion, an essential process involving the fusion of multiple mononuclear cells.Notably, A30L exhibits remarkable similarities to the Vaccinia virus variant Copenhagen A28L [11].Consequently, it is regarded as a promising target for research and development endeavors within the field of monkeypox virus vaccine development.It is imperative for scientists worldwide to enhance their comprehension of this pox virus, encompassing aspects related to prophylactic measures, medicinal diagnosis, and fundamental infection control.This understanding is crucial in order to fully grasp the far-reaching implications of the ongoing outbreak, considering the escalating case numbers observed.
Recent advancements in the field of integrated bioinformatics and immunoinformatics have revolutionized vaccine design, making it faster and more cost-effective.Nowadays, it is possible to design and evaluate the efficacy of targeted immunogenic peptides for their action on biological systems even before the actual vaccine is available for practical experiments.However, traditional methods of vaccine design have their own limitations, such as using the entire organism or large protein residues, which unnecessarily increase the antigenic load and the risk of allergenic reactions [12].We believe that an optimal multi-epitope vaccine can improve immune response and reduce the chances of reinfection by enhancing the host's immunogenicity [13].
In this study, we employed integrated approaches to design a vaccine by identifying B-cell and T-cell, epitopes on the viral protein.Various parameters were considered for this process following the past research.Subsequently, we validated the proposed vaccine construct using in-silico immune simulations by administering the vaccine and monitoring the immune response.In the present study, an immunoinformatics-based approach was devised with the aim of constructing a multi-epitope vaccine targeting monkeypox (Fig. 1).

Protein Sequence Retrieval
The FASTA sequence of the A30L envelope protein derived from Monkeypox virus Zaire-96-I-16, with the NCBI Reference Sequence NP_536567.1,was obtained from the National Center of Biotechnology Information (NCBI) database, accessible at https:// www.ncbi.nlm.nih.gov/.A30L holds significant relevance in the field of monkeypox virus investigation, serving as a vital entity responsible for virus penetration into the host and subsequent cell-cell fusion, manifesting as syncytial formation.

Physiochemical Analysis
The ProtParam server, accessible at (https:// web.expasy.org/ protp aram/) [14], was utilized to predict the physicochemical characteristics of the protein under investigation.Additionally, Protparam was employed to elucidate the fundamental composition of the vaccine.The computational tool ProtParam facilitates the computation of several vital parameters, including the grand average hydropathicity (GRAVY), estimated half-life, molecular weight, instability and aliphatic index, theoretical isoelectric point (pI), amino acid and atomic composition, as well as the extinction coefficient.The GRAVY value of a peptide or protein is determined by summing the hydropathy values of each individual amino acid, dividing the total by the number of residues in the sequence, and subsequently rounding the result to the nearest whole number.

Antigenicity and Toxicity Prediction
To evaluate the immunogenicity of individual epitope fragments, the Vaxijen 2.0 server (http:// www.ddg-pharm fac.net/ vaxij en/ VaxiJ en/ VaxiJ en.html) was employed with a threshold value of 0.5.This server utilizes an alignment-independent approach, estimating antigenicity based on physicochemical characteristics rather than sequence alignment with a prediction accuracy of 70-89% [15].The Vaxijen algorithm employs the auto-cross covariance (ACC) technique to transform protein sequences, generating uniform vectors that encapsulate essential amino acid features.Epitope fragments with a threshold score exceeding 0.4 were identified as potential antigens [16].Furthermore, the toxicity of these epitopes was assessed using the ToxinPred webserver (http:// crdd.osdd.ntheet/ ragha va/ toxin pred/) [17].Epitopes with a half maximal inhibitory concentration (IC50) value lower than 70 nM were selected for further analysis and consideration [18].

Allergenicity Analysis
The allergenicity of the predicted peptides was assessed utilizing the AllerTop 2.0 server (https:// www.ddg-pharm fac.net/ Aller TOP/ index.html).AllerTop employs various machine learning techniques along with amino acid E-descriptors and auto-and cross-covariance transformation of protein sequences into uniform vectors of equal length for the classification of allergies [19].

B-Cell Epitope Prediction
The IEDB (The immune epitope database) server (http:// tools.iedb.org/ main/ bcell/) provides a collection of tools capable of identifying regions within proteins with a high likelihood of being recognized as epitopes in the context of B cell responses.B cells necessitate the activation of their immune response through specific antigens or epitopes in order to produce antibodies that exhibit compositional diversity [20].To predict B-cell epitopes within the Monkeypox virus protein (A30L Monkeypox virus Zaire-96-I-16), the BepiPred 2.0 server (http:// tools.iedb.org/ bcell/ help/# Bepip red-2.0)was employed.BepiPred-2.0 utilizes a Random Forest algorithm trained on non-epitopic and epitopic amino acids derived from crystal structures to identify B-cell epitopes based on sequence information [21].The epitopes with a percentile rank of ≤ 2 and predicted by more than one allele by both methods were considered as strong binders and scrutinized for further analysis [25].

T Cell MHC Class I Binding Predictions
The MHC I binding prediction tool, accessible through the IEDB (http:// tools.iedb.org/ mhci), was employed to forecast the interaction between the investigated sequences and MHC I alleles.This tool incorporates various methods to quantify the binding affinity between specific sequences and designated MHC class I molecule sequences.In this study, an artificial neural network (ANN) approach was employed to determine the half maximal inhibitory concentration (IC50) values for peptide binding to the MHC class I molecule.To facilitate further analysis, alleles displaying binding affinities with IC50 values below 70 nM were selected for subsequent investigation [20,[22][23][24][25].

T Cell MHC Class II Binding Predictions
To predict MHCII peptide binding, the protein sequence was submitted to the IEDB server (http:// tools.iedb.org/ mhcii/) utilizing the NN-align2.3algorithm as the chosen prediction methodology.Peptides with IC50 values below 70 nM were selected for further analysis [26].

Population Coverage
Population coverage analysis is a crucial step to evaluate the effectiveness of the designed multi-epitope vaccine in combating the global monkeypox outbreak across diverse populations.This analysis provides valuable insights into the potential applicability and efficacy of the vaccine in various population groups, aiding in the development of promising vaccines.To assess population coverage, selected MHC class I and II epitopes were examined for their relevance to the worldwide population affected by monkeypox virusrelated mortality and infection.The IEDB population coverage analysis tool (http:// tools.iedb.org/ popul ation/) was utilized with its default parameters for this purpose [27].

Multi-Epitope Vaccine Construction
Epitopes possessing antigenic, non-allergenic, and nontoxic characteristics were isolated and employed in the construction of a multi-epitope vaccine.To facilitate the development of the multi-epitope structure, T-Cell, MHC I, and MHC II epitopes were interconnected using appropriate linkers.An EAAAK linker was utilized to connect the vaccine sequence with the adjuvant sequence.Linkers such as CPGPG were employed to connect B-cell epitopes with MHC I epitopes, while AAY linkers were utilized to connect MHC II epitopes.The adjuvant, derived from Mycobacterium tuberculosis 50S ribosomal protein L7/L12, was incorporated to enhance the immunogenicity of the vaccine.To facilitate protein identification and epitope prediction, a 6xHis tag was appended to the C-terminal end of the vaccine construct [28][29][30].

Prediction of Secondary Structure
The secondary structure of a protein plays a crucial role in determining its folding behavior and physicochemical properties.To predict the secondary structure and evaluate the structural elements and properties of the constructed multiepitope vaccine, we employed the Prabi server (https:// npsaprabi.ibcp.fr/ cgi-bin/ npsa_ autom at.pl? page= npsa_ gor4.html).The server was employed to analyze the secondary structure of the protein including helix, sheet, turns and coil parameters.The server employs GOR IV prediction method to evaluate the sterochemical quality of the protein structure by checking geometry of the residues and the overall structural geometry with a mean accuracy of 64.4% [31].

Disulfide Engineering
To perform disulfide engineering, the DiANNA (DiAminoacid Neural Network Application) webserver (http:// bioin forma tics.bc.edu/ clote lab/ DiANNA/) was employed.This server swiftly evaluated the proximity and geometry of the amino acid residues to identify those suitable for disulfide bond formation [34].

Molecular Docking
Toll-like receptors, commonly referred to as TLRs, are recognition molecules capable of detecting various antigens, leading to the initiation of intracellular signaling pathways.To evaluate the interaction between the multi-epitope vaccine construct and the TLR2 (PDB ID: 6NIG), TLR3 (PDB ID: 2A0Z), TLR4 (PDB ID: 4G8A), TLR5 (PDB ID: 3J0A), and TLR8 (PDB ID: 4R0A) receptors, molecular docking was performed using the HDOCK server (hust.edu.cn)[35,36].This analysis aimed to assess the binding affinity of potential vaccine candidates and their interaction with the receptors.

Molecular Dynamics Simulation
To assess the stability of the Toll-Like receptors and peptide vaccine complex, dynamic studies were conducted.Root mean square deviation (RMSD) and Root mean square fluctuation (RMSF) graphs were generated to monitor the compound's stability.In our investigation, protein-protein dynamic simulations were performed using the GROMACS 2022.4 package [37] with the GROMOS96 54a7 force field.The simulations were executed on a Linux operating system using a command-line interface.A salt concentration of 0.15 M was maintained based on the box size (2 nm) and the number of water molecules.The simulation was run for 50 ns, and the graph ratio was analyzed.Additionally, the Solvent Accessible Surface Area (SASA) was calculated for the last 50 ns to ensure the accuracy of the results.

Equilibration
The equilibration of the complex was performed at a temperature of 310 K and a pressure of 1 bar, utilizing a 2-fs step size.To gradually reach the desired temperature of 310 K, the system was heated using a Berendsen thermostat with a standard constant of 0.1 Ps in the NVT ensemble.Subsequently, the system was pressurized and position restraints were applied in the NVT ensemble using the Parinello-Rahman barostat algorithm [38].

Codon Optimization
Codon optimization is an essential procedure employed to enhance gene expression and improve the translation efficiency of a gene of interest by adapting to the codon bias of the host organism.This is achieved by manipulating the codon usage pattern [39].In this study, codon optimization of the designed multi-epitope vaccine construct was performed using VectorBuilder (https:// rb.gy/ nhhdc3) to enable efficient gene expression in the E. coli (strain K12) host.During the codon optimization process, particular attention was given to avoiding cleavage sites for restriction enzymes.Furthermore, the GC content percentage and Codon Adaptation Index (CAI) results were also obtained to evaluate the effectiveness of the codon optimization strategy.In order to ensure that the vaccine construct has a potential translation, stability and transcription efficiency the ideal CIA values should be greater than 0.8 or close to 1 and the GC content should be in the range of 30 to 70% [25].

Immune Simulation and in silico Cloning
The C-ImmSim web prediction tool (http:// 150.146.2.1/C-IMMSIM/) was employed to simulate the immunogenicity and immune response profile of the designed multi-epitope vaccine construct within the human immune system [40].This server utilizes position-specific scoring matrix (PSSM) and various machine learning algorithms to predict the immune simulation response of the host, that is the human body.In the present study we considered the minimum interval period of 30 days between two clinical doses.We conducted in silico simulations to administer three injections at different time intervals: 1, 84, and 168, corresponding to 8 h per time step in real life and the stimulation parameters were maintained at their default values [25].Furthermore, the modified DNA sequence was incorporated into the pET-28a ( +) vector using the SnapGene in-silico cloning program (version 5.2.3) (www.snapg ene.com).This was accomplished by inserting the vaccine construct between the EcoR1 and BamHI restriction sites positioned at the 5' and 3' ends, respectively [41].

Characterization of A30L Protein Sequence
The A30L protein sequence consists of a total of 146 amino acids, with a molecular weight of 16,403.68kDa.It exhibits stability with an instability score of 29.97.The protein's hydrophobicity is indicated by a grand average hydropathicity (GRAVY) score of 0.057, while the aliphatic index suggests a high proportion of aliphatic residues at 86.78, further supporting its hydrophilic nature.
To predict the antigenicity of the protein, the VaxiJen server was utilized.The threshold for virus proteins is set at 0.4, where scores above 0.5 indicate antigenicity, while scores below 0.5 suggest non-antigenicity with a prediction accuracy of 70-89%.The protein's overall prediction score for antigenicity is 0.6212, surpassing the threshold of 0.4, thus indicating it as a potential antigen.Both the toxinpred server and the allertop server analysis confirm that the protein sequence is non-allergenic and non-toxic, as depicted in Table 1.

B Cell and T Cell Prediction
Epitopes of varying lengths targeting B-cells and T-cells were predicted for the A30L protein derived from the monkeypox virus.Through the implementation of IEDB conservation methods, the BCPRED algorithm specifically identified four epitopes, all exhibiting 100% conservation across peptide sequences and possessing the potential to elicit an immune response (as presented in Table 2).To further evaluate these predicted peptides, bioinformatics web tools were employed to assess their antigenicity, allergenicity, and toxicity profiles.
The peptides, along with their corresponding MHC I and MHC II alleles, were retrieved from the IEDB server.To ensure safety, peptides with IC50 values below 70 were selected, as higher values could pose potential risks.Subsequently, the predicted peptides underwent comprehensive assessments for antigenicity, allergenicity, and toxicity.Optimal vaccine constructs should consist of peptide sequences that exhibit antigenicity (with values ranging from 0.5 to 1.0), while being non-allergenic and non-toxic.Among the tested peptides, a list of 22 peptides successfully met these criteria, making them suitable candidates for vaccine construction.Moreover, these peptides possess the appropriate alleles for both MHC I and MHC II, as illustrated in Table 3.

Population Coverage
The IEDB population coverage analysis tool was utilized to assess the population coverage rate of the predicted epitopes derived from the A30L protein.This analysis involved supplying the promiscuous epitopic core sequences (Class I and Class II) along with their corresponding HLA alleles, as outlined in Table 4.The results of the population coverage analysis are presented in Fig. 2 and 3.The curve in the graph represents the relationship between the number of epitopes included in the vaccine and the population coverage.The PC90 value is the minimum number of epitope hits/HLA combinations recognized by 90% of the population.In other words, it is the minimum number of epitopes that need to be included in a vaccine to ensure that 90% of the population will have an immune response to it.The PC90 values for MHC Class I and II were 0.32 and 0.23 respectively.

Construction of Vaccines and Their Physiochemical Properties
The vaccine was constructed using the adjuvant and linkers connecting the B cell and T cell predicted peptides, which possess desired characteristics of being Antigenic, non-allergenic, and non-toxic.The resulting vaccine has a predicted molecular weight of 55,692.38 kDa.Its theoretical  human reticulocytes in vitro is 30 h, while it exceeds 20 h in yeast in vivo and 10 h in E. coli in vivo.These properties are depicted in Fig. 4.

Antigenicity, Allergenicity, and Toxicity Prediction of Vaccine Construct
The multi-epitope vaccine construct underwent thorough evaluation for its Antigenicity, allergenicity, and toxicity using reputable bioinformatics online tools including Vaxigen, allertop, and toxinpred.The results of the analysis revealed that the constructed vaccine exhibited a high degree of Antigenicity, with a score of 0.6286 surpassing the threshold value of 0.4.Furthermore, the assessment demonstrated that the vaccine does not pose any allergenic risks nor exhibit any toxic properties.Based on these findings, it can be concluded that the multi-epitope vaccine is suitable for utilization as a preventive measure against the targeted disease and requires biological validation in experimental models.

Secondary Structure Prediction
The Psipred server was employed to conduct a computational analysis aiming to ascertain the distribution of secondary structure elements within the multi-epitope vaccine.The anticipated structural composition of the vaccine encompassed an alpha-helix component, accounting for 55.74% of the structure, represented by a count of 291 residues.Additionally, a beta-strand component constituted 9.19% of the structure, consisting of 48 residues.Finally, a significant portion of the structure, approximately 35.05%, was attributed to the random coil conformation, comprising 183 residues.The Psipred server provided a comprehensive sequence plot and a visually appealing cartoon depiction of the secondary structure arrangement (Fig. 5).

Tertiary Structure Prediction of the Vaccine Construct
The I-TASSER server was employed to predict the tertiary structure of the multi-epitope constructed vaccine.Five models were generated, each with its corresponding C-score ranging from -1.00 to -4.29.Typically, C-scores ranging between -5 and -2 were poor, with higher values indicating greater confidence in the predicted structure [27,28].Among the five models, the one with the highest C-score was selected for further investigation and analysis (Fig. 6).
To assess the quality of the selected model, a Ramachandran plot analysis was performed.The analysis revealed that 97.6% of the amino acids in the vaccine construct model were located within the most favoured regions, indicating a high-quality structure.Only 2.4% of the amino acids were found in the disallowed region, suggesting that the model's overall conformation was favourable and consistent with protein folding principles (Fig. 7).

Molecular Docking
Using the H Dock server, molecular docking between the designed vaccine and the TLRs (TLR2, TLR3, TLR4, TLR5, and TLR8) was performed.The first model in each docking that was predicted by the HDOCK server was selected for further study (Fig. 8).The docking scores for the TLR2-, TLR3-, TLR4-, TLR5-, and TLR8-vaccines were respectively -287.79, -408.81,-369.01,-361.81, and -363.03.It demonstrates that as compared to other TLRs, the TLR3 and TLR4 have a higher docking score.

Molecular Dynamic Simulation
The molecular dynamic simulation was conducted to assess the interaction between the best-docked Toll-like receptors (TLRs) and our designed peptide structure.The simulation process involved several steps, starting with energy minimization using the steepest Descent algorithm.This was followed by NVT (constant Number of particles, Volume, and Temperature) and NPT (constant Number of particles, Pressure, and Temperature) equilibration steps, each consisting of 50,000 simulation steps.
The simulation was performed for a duration of 50 ns to evaluate the stability of the TLR-peptide complexes and analyze solvent accessibility.Various parameters, including Root Mean Square Deviation (RMSD), Radius of Gyration (RG), and Solvent Accessible Surface Area (SASA), were calculated based on the simulation data.It was observed that the complexes exhibited greater stability during the last 30 ns of the simulation.
Specifically, TLR2, TLR3, and TLR5 showed notable stability throughout the entire 50 ns of the simulation, as depicted in Figs. 9, 10 and 11.These findings suggest favorable interactions and structural stability between the TLRs and the designed peptide structure.

Codon Optimization
To maximize the amount of protein that can be expressed from the vaccine construct in E. coli (strain K12), codon optimization was performed.The length of the codon sequence that was optimized was 1569 nucleotides.The optimized nucleotide sequence had a CAI of 0.92, which means it was made up of the most commonly occurring codons.The optimized sequence has a GC content of 51.82% on average.The higher the GC concentration, the greater the protein expression in prokaryotes.Employing the SnapGene tool, the nucleotide sequence that had been optimized was cloned into the pET28a ( +) vector (Fig. 12).In the present study pET28a + was employed as the expression vector as they proffer high level of expression, multiple cloning sites, N-terminal HIS tags, smaller size, and compatibility with the bacterial strains.The multiple cloning site of pET28a + enables easy gene insertion and the smaller size eliminates the need for larger inserts.Additionally, the N-terminal 6 × HIS tags facilitate easy purification of the protein through nickel-affinity chromatography and this vector is compatible with commonly used bacterial strains.The inclusion and exclusion of the protein transmembrane region predominantly depends on the nature of the study.Generally, the transmembrane regions region portrays an indispensable role in localization and function.When the study objective is to elucidate the function of the protein within the cellular membrane, the transmembrane region is included in the investigation as this will ensure that the expressed protein is properly targeted to the membrane and they carry out their normal functions.Similarly, when the extracellular and intracellular domains of the protein is investigated the transmembrane regions are commonly excluded by truncating the protein at the transmembrane region by using the deletion mutants which lack the transmembrane region.In the present research the transmembrane region was included as the function of A30L protein was investigated [42].

In-silico Immune Simulation
The in-silico immune simulation was investigated by employing the ImmSim simulator (Fig. 13).The graphs displayed that the all the primary immune response including Ag levels, IgM, IgG,and IgG1 + IgG2 were induced from approximately 0-15 days (Fig. 13 a).It was noticed that the Ag levels decreased after ~ 5 days after which it remained stable for the following days.Following this, it was noticed that the IgM, IgG, IgG1 + IgG2 levels significantly increased from ~ 10 to ~ 18 days (Fig. 13a).The vaccine construct demonstrated lower Simpson index (D), indicating higher diversity of the vaccine (Fig. 13b).Additionally, they also displayed higher response of T-helper and T-cytotoxic cells and the significant development of memory cells were observed (Fig. 13c).In addition to these, several B-cells and B-memory cells were generated and there was a notable presentation of B-cell isotype IgM (Fig. 13d).

Discussion
Monkeypox, a zoonotic viral illness, exhibits similarities to past smallpox outbreaks but generally presents with less severe symptoms.It is caused by the monkeypox virus and primarily affects regions in Africa, posing a significant risk to human health [1,5,10].However, recent cases have been reported in other parts of the world, indicating its potential to become a global public health concern within a short span of time.Presently, there is no targeted treatment available for monkeypox [5,7].Supportive care, which focuses on  7 Ramachandran plot of the vaccine construct (Model 1) using PROCHECK.The secondary structure validation of the vaccine construct revealed the model had a high-quality structure, with 97.6% of amino acids in favoured regions and only 2.4% in disallowed regions, indicating favourable conformation and adherence to protein folding principles.Additionally, the construct did not exhibit any stearic hindrance or bad angle and the Z score analysis (Z Score = -1.2) indicates that the overall quality of the modelled structure was good symptom management and prevention of secondary infections, constitutes the main approach.This may involve strategies such as maintaining hydration, ensuring adequate rest, and treating any concurrent infections that may arise due to the disease [10].A significant challenge in managing monkeypox lies in the absence of a specific antiviral drug.Unlike smallpox, which was eradicated through global vaccination efforts, there is currently no dedicated vaccine for monkeypox, and the smallpox vaccine does not confer complete protection against it.Ongoing research aims to develop a specific vaccine for monkeypox, but this endeavor is complex and demanding.Furthermore, effective treatments targeting the underlying virus are lacking in current therapies for monkeypox.While supportive care can alleviate symptoms, it does not directly address the root cause of the disease.Severe cases of monkeypox may lead to complications such as pneumonia, encephalitis, and sepsis, for which specific treatments are presently unavailable [43].Multiepitope vaccines represent a promising strategy for addressing monkeypox virus diseases.Instead of targeting a single antigenic site, these vaccines are designed to recognize multiple epitopes on the virus [44].This approach offers the advantage of eliciting a more comprehensive and longlasting immune response, thereby increasing the potential for effective disease protection.Preclinical studies have demonstrated the immunogenicity of multiepitope vaccines against monkeypox, and some of these vaccines have progressed to early-phase clinical trials [45].Notably, these clinical trials have shown favorable tolerability and robust immune responses among participants, indicating the potential effectiveness of multiepitope vaccines in safeguarding against monkeypox.A significant benefit of multiepitope vaccines lies in their ability to target multiple epitopes, enhancing the durability of the immune response.This is particularly crucial for monkeypox, as the virus exhibits rapid evolution and alterations in its antigenic profile, making it challenging to combat with a single-epitope vaccine.By engaging multiple epitopes, a multiepitope vaccine can ensure sustained efficacy, even in the face of viral evolution.Additionally, multiepitope vaccines have the capacity to elicit a broad and potent immune response encompassing both cellular and humoral immunity.This comprehensive immune activation enables the vaccine to effectively target diverse virus strains and potential future variants that may arise [44,46].
In this study, a multi-epitope subunit vaccine was developed through in-silico screenings.The FASTA sequence of the A30L envelope protein from Monkeypox virus Zaire-96-I-16 was obtained from the NCBI database and subjected to sequence analysis using the ProtParam server.The analysis revealed an antigenicity score of 0.6212, indicating the potential of these proteins as antigens.Furthermore, they exhibited non-toxic characteristics, as indicated in Table 1.The objective of this vaccine is to elicit an immune response against MPXV by targeting the cell envelope protein A30L.The primary aim is to design a novel vaccine that ensures complete safety with minimal adverse effects.Hence, in this study, only vaccines demonstrating minimal and negligible infectivity were selected.The envelope protein plays a critical role in various essential biological activities of MPXV, including host cell entry, cell-cell fusion, and interaction between the virus and the host.The A30L protein, encoded by the monkeypox virus, serves as a crucial component in the pathogenesis of the disease.Belonging to the ankyrin repeat protein family, this viral protein is implicated in diverse cellular processes, including gene expression regulation and immune response modulation.Investigations have established the significance of the A30L protein in facilitating viral replication and dissemination within infected cells.It achieves this by interacting with cellular proteins and manipulating the cellular milieu, thereby enhancing the virus's replication and spread.Furthermore, the A30L protein exerts influence over the immune response, potentially influencing the disease's severity.A prominent mechanism through which the A30L protein contributes to monkeypox pathogenesis involves its interference with the interferon signaling pathway.This pathway constitutes a critical aspect of the host's antiviral defense, and the A30L protein disrupts its functioning, potentially facilitating viral persistence and dissemination.Additionally, the A30L protein engages in interactions with cellular proteins and induces modifications, subsequently impacting their functionality and influencing disease progression.For instance, the A30L protein has been observed to interact with immune response-regulating cellular proteins, potentially contributing to the immunosuppressive state observed in certain monkeypox patients [47].
The findings of this study indicate that for the vaccine to achieve global acceptance, the provided epitopes should be capable of binding to a wide range of ethnically diverse MHC I and MHC II alleles, with high population coverage scores.To evaluate the population coverage of the predicted T lymphocyte-reacting epitopes, an analysis was conducted.The predicted epitopes demonstrated significant associations with various MHC I and MHC II alleles, thus encompassing diverse sets of alleles with high population coverage scores.The envelope polyprotein contains numerous well-known epitopes that engage with MHC I and MHC II alleles in T lymphocytes.Consequently, peptides exhibiting antigenicity, non-allergenicity, and non-toxicity were meticulously chosen as potential vaccine candidates.The results of this study indicate that the proposed epitopes for vaccination have the potential to efficiently interact with common human alleles worldwide, The prediction of MHC class I epitopes is a crucial step in the development of vaccines.In this study, the specific viral components of MPXV were subjected to various web-based MHC I and II epitope prediction tools.The T cell epitopes were then internally evaluated based on established criteria before being selected for further investigation.Two main selection criteria were considered: the candidate antigen(s) should exhibit sufficient immunogenicity, and they should also be accessible to membrane-bound or free antibodies.Hence, IC50 values lower than 70, which indicate significant immunogenicity, were used as a cutoff for selection.Based on promising results, the construction of a vaccine using the proposed peptides appears to be a high priority, with the potential for widespread deployment as a universal epitopebased peptide vaccine against MPXV.The humoral immune response mediated by B-lymphocytes plays a critical role in combating monkeypox virus antigens, as it generates a diverse repertoire of pathogen-specific antibodies that ultimately contribute to the reduction of viral load.To predict the B-cell epitope of the Monkeypox virus protein (A30L Monkeypox virus Zaire-96-I-16) for incorporation into the vaccine design, the BCPRED service of the IEDB server was utilized.Four conserved epitopes with significant scores were identified, further supporting their potential inclusion in the vaccine formulation.
The selection of B-cell and T-cell epitopes was based on various factors such as antigenicity, allergenicity, and toxicity.The B-cell epitopes predicted by the IEDB were characterized by their flexibility and hydrophilicity.These predicted peptides ranged in length from 7 to 43 amino acids.Among the four predicted peptides, the peptide spanning from 30 to 72 exhibited antigenicity, while the remaining peptides were non-antigenic.All the predicted B-cell peptides were found to be non-toxic, except for the peptide spanning from 130 to 138, which displayed allergenicity.This suggests a limited number of favorable interactions Fig. 12 In silico cloning of the multi-epitope vaccine into the pET28a ( +) vector using SnapGene software free-trial (https:// www.snapg ene.com/ free-trial/).The red section represents the vaccine construct and the black section shows the backbone of the vector between B-cells and MPXV.Furthermore, it is important to note that while the humoral response from memory B cells can be overridden over time by various antigens, cellmediated immunity often results in long-lasting protection.In this study, T-cell MHC I and MHC II epitopes were linked together using AAY linkers, whereas MHC I and B-cell epitopes were linked using CPGPG linkers.These linkers not only facilitate epitope presentation but also prevent the formation of junctional epitopes.Additionally, the EAAAK linker was used to enhance stability and reduce linkage with other protein regions.To improve the vaccine's immunogenicity, an adjuvant derived from Mycobacterium tuberculosis, specifically the 50S ribosomal protein L7/L12, was employed [48].
To generate structural templates, the I-TASSER algorithm employed a multiple-threading technique utilizing LOM-ETS (Local Meta-Threading Server, version 3).Full-length atomic models were then constructed through iterative simulations of fragment assembly based on these templates.The accuracy of the predictive model was evaluated using the C-score.To investigate the binding affinity of the vaccine construct with TLR proteins, molecular docking was conducted using the HDOCK server.The docking scores for the vaccine complexes with TLR4, TLR5, and TLR8 exhibited similar values, indicating comparable binding affinities.
The docking analysis revealed that the TLR3-vaccine construct complex exhibits the highest predicted docking score among all the complexes tested.TLR3, a pivotal protein in the immune system, plays a critical role in mounting an immune response against viral infections.As a pattern recognition receptor (PRR), TLR3 specifically recognizes viral double-stranded RNA (dsRNA), a common molecular pattern found in numerous viruses.Upon recognition of dsRNA, TLR3 initiates signaling pathways that trigger the production of cytokines and interferons, crucial molecules involved in immune defense against viruses [49,50].These signaling molecules serve to alert other immune cells to the presence of the virus, activating them to coordinate a concerted response aimed at eliminating the viral threat.Hence, TLR3 assumes a vital role as a fundamental component of the innate immune system's defense against viral infections and is instrumental in generating an effective antiviral immune response.For subsequent expression and purification, the vaccine construct was successfully cloned into the appropriate vector.The pET28a ( +) vector is widely recognized for its suitability in protein expression endeavors [50].
To mitigate the issue of codon biases that often arise when expressing eukaryotic proteins in prokaryotic vectors, codon optimization was implemented as a precautionary measure.Achieving optimal expression of the vaccine design necessitated employing a suitable Escherichia coli (E.coli) expression vector [51,52].Prior to cloning into the pET28a ( +) vector, the target vaccine construct was subjected to reverse transcription and modifications tailored for compatibility with the E. coli K12 strain.With a codon adaptation index of 0.92 and a GC content of 51.82%, it was anticipated that the vaccine construct would be expressed at high levels in the bacterial host.These two parameters serve as indicators of the vector's capacity to facilitate robust expression of the vaccine construct.Subsequently, the gene encoding the vaccine construct was cloned into the vector, employing various cloning sites, successfully incorporating the vaccination protein into the vector [52].
Previously, there have been many research conducted to find suitable multi-epitope vaccines various proteins implicated in the pathogenesis of MPXV while some of them targeted the genome of the pox virus [53][54][55][56].A study by Shantier et.al, developed an antigenic 275 amnio acid multiepitope vaccine with greater solubility against cell surface protein [53].Saifullah et.al., designed multi-epitope-based vaccine against the surface proteins MPXVgp002 and MPX-Vgp008 with 99.74% population coverage worldwide [57].Asad et al., developed the vaccines for extracellular proteins including, cupin domain-containing protein, ABC transporter ATP-binding protein and DUF192 domain-containing protein [58].All of the above-mentioned research has implemented methods as in the present research and the results were comparable.The common characteristic was of the vaccine found to be potentially effective against MPXV and to be highly immunogenic, cytokine-producing, antigenic, non-toxic, non-allergenic, and stable.
A study designed a universal vaccine for Monkeypox, Smallpox and Vaccinia Viruses by targeting A29, A30, A35, B6 and M1 antigens to prevent the pandemics [59].A study by Hamid et.al, targeted the monkeypox antigenic proteins E8L, A30L, A35R, A29L, and B21R and reported two multiepitope vaccine candidates, ALALAR and ALAL as suitable vaccine candidates against MPXV [60].It was observed that A30L was studied as a common protein for other viral diseases and there were no studies reporting multi-epitope vaccines designed especially for MPXV [60][61][62].The present study reports that the peptides ATHAAFEYSK, FFIV-VATAAV, and MNSLSIFFV were antigenic, non-allergen and non-toxic in nature.Though there are numerous studies that has tried to design multi-epitope vaccine against various MPXV, it is important to note that monkeypox virus has high mutation rate, which can eventually lead to changes in the epitopes targeted by the vaccines thereby reducing its effectiveness.Additionally, limited information on the epitopes that are located in the conserved regions across different strains of monkeypox, makes it challenging to design a vaccine that provides broad protection.These problems could be addressed by identifying conserved epitopes or by the incorporating the adjuvants to the vaccine construct, its effectiveness could be increased.

Conclusion
Despite the severity of the MPXV disease and its widespread occurrence, an effective therapeutic vaccine against MPXV is currently unavailable.The recent outbreak of MPXV has highlighted the urgent need for a reliable vaccine to combat this formidable pathogen.In this study, we have employed advanced immunoinformatic techniques and in silico methods to design a multi-epitope vaccine capable of eliciting a robust immune response against MPXV.The A30L envelope protein of MKXP has been identified as a promising target for developing this innovative vaccine, which aims to ensure safety and minimize adverse effects.By identifying both B-cell and T-cell epitopes within the A30L protein, we have selected epitopes with optimal antigenicity, while carefully evaluating their toxicity and allergenicity.The chosen epitopes were strategically linked and supplemented with suitable adjuvants to enhance their immunogenicity.The designed vaccine exhibited favorable characteristics, including non-allergenicity and antigenicity, as well as satisfactory physicochemical properties.Through a comprehensive in silico approach, this study successfully identified a promising multi-epitope design.These findings lay the foundation for further investigation through in vitro and in vivo experiments, facilitating the development of effective therapies and serving as a driving force for future MPXV vaccine development.
However, multiepitope vaccine design faces several inherent limitations, primarily stemming from the challenge of accurately predicting the optimal combination and arrangement of epitopes to elicit an effective immune response.To validate the theoretical predictions, experimental investigation becomes imperative.However, conducting in vitro studies presents hurdles in replicating the intricate in vivo environment, particularly the intricate interplay between immune cells, antigen-presenting cells, and the targeted pathogen.In vivo studies also encounter obstacles, including the possibility of adverse effects, scarcity of suitable animal models, and the complexity of accurately measuring immune responses.Consequently, both in vitro and in vivo studies are indispensable for evaluating the safety and efficacy of multiepitope vaccines before their clinical implementation.
Acknowledgements The authors would like to acknowledge the Bioinformatics lab facility, BioNome (https:// biono me.in/), Bangalore, 560043, India, for their assistance in the work.The help from Dr. Sameer Sharma and his team, Bioinformatics lab, was greatly helpful in performing MD simulation analysis via Gromacs v2019.4.

Authors Contribution
All the authors have equally contributed to the manuscript.
Funding Not Applicable.

Fig. 1 A
Fig. 1 A schematic illustration of the immunoinformatic approaches used to design a multi-epitope vaccine

Fig. 5 A
Fig. 5 A Sequence plot B Psipred Cartoon

Fig. 6
Fig. 6 Top Five Tertiary Structure Models of Vaccine Construct

Fig. 13
Fig. 13 ImmSim presentation of an in silico immune simulation with the multi-epitope vaccine construct.a Immunoglobulin production in response to antigen injection, b B-cell populations after antigen injec-

Table 1
Assessing Antigenicity, allergenicity, and toxicity of A30L Protein Sequence pathicity (GRAVY) score is projected to be 0.338, signifying a hydrophilic nature.Additionally, the calculated aliphatic index of 92.66 further supports the protein's hydrophilic characteristics.The estimated half-life of the vaccine in

Table 3
MHC I and MHC II peptide prediction