Engineering a broad-spectrum multi-epitope vaccine to combat emerging monkeypox virus by immunoinformatic approaches

doi:10.21203/rs.3.rs-4908990/v1

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Engineering a broad-spectrum multi-epitope vaccine to combat emerging monkeypox virus by immunoinformatic approaches

https://doi.org/10.21203/rs.3.rs-4908990/v1

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Monkeypox virus (MPXV), has caused 41,664 confirmed cases and five deaths in non-endemic regions, as reported by the World Health Organization (WHO). There is an urgent demand for effective vaccines to combat and prevent the spread of MPXV. Traditional vaccine development is low-throughput, expensive, time-consuming, and susceptible to reversion to virulence. As an alternative, a reverse vaccinology approach can be employed as a promising tool to design effective and safe vaccines against MPXV. Here, MPXV proteins associated with viral infection were analyzed for potential immunogenic epitopes to design multi-epitope vaccine constructs based on B-cell, CD4+, and CD8+ epitopes. Epitopes were selected based on allergenicity, antigenicity, and toxicity parameters. The prioritized epitopes were then combined via peptide linkers and N-terminally fused to various protein adjuvants, including PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and the cholera toxin B subunit (CTB). All vaccine constructs were further computationally validated for physicochemical properties, antigenicity potential, allergenicity, safety, solubility, and structural stability. The three-dimensional structure of the selected construct was also predicted. Moreover, molecular docking and molecular dynamics (MD) simulations between the vaccine and the TLR-4 immune receptor demonstrated a strong and stable interaction. The vaccine construct was codon-optimized for high expression in the E. coli platform and was finally cloned in silico into the pET21a(+) vector. Collectively, these results could represent innovative tools for vaccine formulation against MPXV and be transformative for other infectious diseases.

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Health sciences/Molecular medicine

Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV) which is an enveloped double-stranded DNA (dsDNA) virus which is categorized in the Orthopoxvirus genus of the Poxviridae family. The symptoms of monkeypox include fever, rash, lymphadenopathy and intense asthenia; however, in certain groups (children, pregnant women, and immunocompromised people) might suffer from serious complications, including secondary bacterial infections, pneumonitis, respiratory distress, sepsis, encephalitis and loss of vision due to cornea infection. The fatality rate of MPX ranges between 0 and 11% with a self-limiting disease lasting 2–4 weeks ¹. The MPXV was first discovered in monkeys in 1970; however, it was first confirmed in human in 1970 in a 9-month-old child with suspected smallpox in the Democratic Republic of Congo ². Thereafter, infection cases have been occasionally reported, but most of MPX cases were restricted in West and Central African nations with a mortality rate between 1 and 10% ^3,4. Monkeypox was documented to be transmitted by travelers from human-to-human transmission or by exposure to imported animals⁵. Since early May 2022, the global outbreak of monkeypox (MPX) disease by monkeypox virus has dramatically increased in the number of MPXV infections worldwide with a total of 92,783 laboratory-confirmed infection cases with 171 deaths reported by World Health Organization (WHO) from 116 reporting countries (as of 19^th December 2023) ⁶. Given the widespread of MPX disease, WHO has declared MPX disease a public health emergency of international concern in July 2022. Due to genetic similarity to smallpox, patients with severe MPXV infection were treated with antiviral medicines for smallpox virus infection, including tecovirimat, cidofovir, and brincidofovir, are currently used to treat MPX under certain conditions; however, high-dose antiviral drug medication tend to cause hepatic malfunctions, while low-dose treatment may lead to the risks of relapse ⁷. Thus, there is an urgent demand for effective vaccines against MPXV infection to curb the number of cases.

However, generating such vaccines remains a significant challenge and to date, there is no specialized vaccines developed specifically to protect against MPX infection. Several smallpox vaccines, includes ACM2000, MVA-BN, and LC16 are available for MPX vaccination based on different jurisdictions. The US-FDA approved ACAM2000 is available for smallpox and MPX vaccination. However, ACAM2000 is a live, replication-competent Vaccinia virus, there is a risk of side effects like injection site pain, lymph node pain, pruritus, and other flu-like symptoms. Additionally, serious and long-term side effects, including myopericarditis or pericarditis have been reported in every 20 cases out of 100,000 ACAM2000 vaccine recipients ⁸. MVA-BN, also known as JYNNEOS, a non-replicating modified Vaccinia Ankara virus vaccine, is an approved by the US FDA in 2019 for the prevention of both smallpox and monkeypox in the United States. Since Jynneos is a replication-deficient live virus vaccine, it is safer than ACAM2000, in particular for vaccination in immunocompromised individuals, including like transplant recipients, patients with HIV, and atopic dermatitis ^9,10. However, limited data on the efficacy of Jynneos against MPXV infection in humans has been documented ¹¹. LC16, third-generation smallpox vaccine, is a live, replicating attenuated vaccines derived from the Lister (Elstree) strain of vaccinia and is approved for the prevention of MPX in August 2000 in Japan ^12,13. Although preclinical evidence demonstrated a safety profile in animal models, LC16 vaccine is not recommended for administration among immunocompromised and atopic dermatitis patients ^8,14,15. Nevertheless, the efficacy of these vaccines against monkeypox is contingent on data derived from animal studies demonstrating immune protection against the monkeypox virus in non-human primates rather than human subjects ¹⁶.

MPXV demonstrated high mutation rate which is 6-12 folds higher than previously expected and some mutations lead to higher transmissible and immune evasion. Thus, there is an urgent demand the development of novel vaccine design methods ^12,17. The drawbacks of conventional vaccine development are low-throughput, expensive, and time-consuming, demanding the culture of pathogens and the identification of their immunogenic components^18,19. To address these shortcomings of the conventional vaccine development, reverse vaccinology approaches have revolutionized vaccine design, and thus enabling scientists to identify immunogenic components and highly conserved epitopes from genome sequence and proteome information without the requirement to isolate and culture the targeted pathogen, and thus offering savings in time and resource²⁰. Multi-epitope vaccines developed by immunoinformatic approach has demonstrated robust immune protection against microbial infection because of fewer side effects, higher specificity, more safety, low-cost production than conventional vaccines, and simultaneous induction of diverse immune responses, including innate, cellular and humoral immune responses^21,22. To develop effective multi-epitope vaccine, the critical step is target selection. Since there is no report on specific MPXV proteins dominantly implicated in MPXV infection, several proteins, including structural and non-structural proteins could play a critical role in the infection by MPXV and could be promising targets for vaccine development. Some MPXV proteins such as M1R (Myristylprotein), B6R (EEV type-I membrane glycoprotein), and H3L (IMV heparin binding surface protein) have been associated with immune responses within poxviruses and, accordingly, are considered as attractive targets for vaccine development²³. In addition, B9R has been reported to involve in pathogenesis of MPXV²⁴. F8L (DNA polymerase) has been previously reported to stimulate the CD8+ T cell population and the release of interferon-g in rhesus macaques²⁵. Accordingly, both structural proteins and non-structural proteins can be promising targets for vaccine development.

Here, a panel of multi-epitope vaccines were designed against the MPXV using a reverse vaccinology approach. MPXV proteins were retrieved from the GenBank database and filtered through several criteria to select final candidates for vaccine construction. Both structural proteins and non-structural proteins were selected as the protein targets for multi-epitope vaccine development. According to previous studies, B6R, H3L, and F8L can stimulate interferon-γ production and activate cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) ^26–30. E4R has been reported to induce antibodies in vivo among prairie dogs ³¹. A40L, B9R, and C2L are homologous to A38L, B8R, and K2L vaccinia viral protein reported to be crucial ligands to MHC class II and HTL ^32,33. J3R/J1l plays a pivotal role in immuno-modulating and preventing apoptosis in host cell, and thus facilitating viral entry and infection ³⁴. Moreover, to provide broad-spectrum protection, six different MPXV strains were employed as our research objects. All candidate epitopes were then further selected based on antigenicity, low allergenicity, and low toxicity. To enhance immuno-stimulation, an adjuvant protein sequence was fused to the N-terminal of each vaccine construct. Collectively, our vaccine candidate could be able to bind and trigger immune response via TLR-4 based on molecular docking and molecular dynamic simulation. Hence, our MPXV multi-epitope vaccine constructs could be a viable alternative to prevent the global outbreak of MPX pandemic.

Monkeypox viral proteins retrieval and screening. Monkeypox protein sequences, including structural and non-structural proteins retrieved from MPXV strain Congo_2003_358 on NCBI database (DQ011154.1) were predicted for antigenicity and allergenicity via VaxiJen 2.0 and AllergenFP1.0. From 200 proteins retrieved from the completed genome, only 24 proteins were selected based on non-allergenicity and antigenicity with a score higher than 0.4 as demonstrated in Table 1A and 1B. These selected 24 proteins were further utilized for epitope prediction.

Prediction of epitope candidates and assessment of allergenicity, antigenicity, and toxicity parameters.NetMHCpan 4.1 BA and NetMHCpan 4.1 EL server predicted 35 CTL epitopes which demonstrated strong binding affinity on MHC class I for HLA Class I alleles (HLA-A0101, HLA A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702, HLA-B0801, HLA-B2705, HLA-B3901, HLA-B4001, and HLA-B5801). All selected 35 CTL epitopes demonstrated positive immunogenicity score via IEDB Class I immunogenicity tool, antigenicity with a score higher than 0.4, non-allergenicity, and non-toxicity.

HTL epitopes were predicted through NetMHCpanII 4.1 BA and NetMHCpanII 4.1 EL for MHC class II. These servers predicted 24 potential HTL epitopes with strong binding affinity against MHC class II based on HLA alleles DRB1_0101, DRB1_0301, DRB1_0401, DRB1_0701, DRB1_0801, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1201, DRB1_1301, DRB1_1501, and DRB1_1602. Additionally, those HTL epitope candidates were tested with IFNepitope server to selectively filter those sequences that can stimulate IFN-g. All 24 HTL epitopes could trigger IFN-g secretion. Out of 24 epitopes, only one epitope showed low antigenicity score (less than 0.4) and thus 23 HTL epitopes were reserved with high antigenicity, non-allergenicity, and non-toxicity

In case of LBL, BepiPred Linear Epitope Prediction 2.0 server was deployed to predict the LBL epitopes with the default threshold of 0.5. The server yielded a total of 30 epitopes predicted to show high antigenic score, non-allergenicity, and non-toxicity. The repertoires of CTL, HTL, and LBL epitopes were utilized for further analysis to select final candidates for vaccine construction.

Transmembrane and signal peptide screening. CTL, HTL, and LBL predicted epitopes obtained from previous experiments were further screened for transmembrane region and signal peptide by utilizing DeepTMHMM and SignalP 6.0. DeepTMHMM was used to predict transmembrane regions of epitopes which should be removed to avoid hydrophobic effect that is prone to aggregate, and thus leading to low-yielding and unstable protein synthesis ^35–37 (Supplementary Dataset 1). CTL, HTL, and LBL epitopes demonstrating potential transmembrane region were retracted from vaccine construction. Our CTL, HTL, and LBL epitopes contained no transmembrane region. Finally, SignalP 6.0 was used to predict the signal peptide regions of CTL, HTL, and LBL epitopes and no signal peptides were found in our CTL, HTL, and LBL epitopes.

Epitope conservancy for the board spectrum coverage of vaccine over different clades. To generate broad spectrum vaccine against various clades of Monkeypox virus, all predicted CTL, HTL, and LBL epitopes were analyzed for the epitope conservancy across 7 clades of Monkeypox virus. Nearly all epitopes could cover Clade I (Zaire-96-l-16), Clade IIa (USA 2003), Clade llb-A (Nigeria 2018), Clade IIb-A.2 (USA 2002), Clade IIb-A.1 (UK P2), Clade IIb-A.1.1 (USA2021), Clade IIb-B.1(France 2022) with 100% of conservancy. However, a HTL epitope derived from F8L protein (SMVFEYRASTVIKGP) showed only a single amino acid variation from valine to isoleucine. One valine in Clade I Congo_2003_358 and Zaire-96-l-16 was replaced by isoleucine in Clade IIa (USA 2003), Clade llb-A (Nigeria 2018), Clade IIb-A.2 (USA 2002), Clade IIb-A.1 (UK P2), Clade IIb-A.1.1 (USA2021), and Clade IIb-B.1(France 2022). In other words, SMVFEYRASTVIKGP in Clade I became SMVFEYRASTIIKGP in Clade II (Supplementary S1 Table). Considering amino acid property, both valine and isoleucine are categorized in hydrophobic amino acid group, and thus valine to isoleucine substitution should produce negligible effect to the epitope.

Epitopes homologous analysis with human proteome. To prevent autoimmune induced by epitopes, Peptide Search server from Uniprot database was employed to investigate epitopes which showed homologous sequences to human proteome. According to Peptide Match tool, predicted epitopes were checked against homo sapiens proteome (Taxonomy ID: 9606). The results found that all epitopes were not homologous to the human proteome.

Final epitopes selection and worldwide population coverage. Epitopes showing non-homologous sequence to human proteome were further selected for the vaccine construct. To ensure broad HLA coverage, epitopes were chosen to target different alleles for both CTL and HTL. These candidates were selectively recruited, based on HLA alleles coverage for CTL and HTL epitopes, and high antigenicity for all three types (CTL, HTL, LBL). Following this method, 9 CTL, 4 HTL, and 4 LBL epitopes were selected as illustrated in Table 2A, 2B and 2C.

Afterwards, those selected CTL and HTL epitopes were analyzed by IEDB population coverage tool. The distribution of HLA alleles targeted by predicted MHC class I and II epitopes combined. The results showed that they could cover 94.87% of world population across 115 nations and 21 ethnicities within 16 geographic regions as shown in Fig. 1.

Multi-epitope subunit vaccine construct. The design of vaccine construct was formulated by combination of 5 elements, including adjuvant, 9 CTL epitopes, 4 HTL epitopes, 4 LBL epitopes, and 6xHis tag, with the help of linkers, as illustrated in Fig. 2. Linkers have been reported to provide stability to vaccine construct and prevent cross-interruption of each epitope, thus rendering them to independently function upon vaccination ^38,39. Five different adjuvants (Cholera toxin B subunit (CTB), PADRE, Beta-defensin 3, 50S ribosomal protein L7/12, and RS-09) were used in this study, resulting in five vaccine constructs. Each adjuvant was N- terminally fused to the vaccine by using EAAAK linker. The CTL epitopes were linked with AAY linkers and GPGPG linkers were used to connect the HTL epitopes. The B-cell epitopes were linked with the help of KK linkers. HEYGAEALERAG linkers were used to connect the first HTL epitope and the last CTL epitope as well linking the first B-cell-epitopes the last HTL epitope. Additionally, 6xHis tag was fused to the C-terminal of the vaccine construct by the RVRR linker for vaccine purification by affinity chromatography and immunodetection of vaccine expression. The schematic of each vaccine construct was depicted in Fig. 2. All five vaccine constructs were subjected to be predicted for antigenicity, allergenicity, toxicity, solubility, the presence of signal peptide, and physicochemical properties compared to a control vaccine without adjuvant.

Antigenicity, Allergenicity, Toxicity and Physicochemical properties. Five vaccines constructs were evaluated and compared for antigenicity, allergenicity, toxicity, and physicochemical properties as shown in Table 3. All five vaccine constructs demonstrated antigenicity score higher than 0.50 calculated by Vaxijen 2.0 server which indicated robust antigenic property of all multi-epitope vaccine constructs. Additionally, four vaccines were immunogenic and non-toxic; however, the vaccine with beta-defensin adjuvant was toxic. The physicochemical properties predicted from ProtParam server showed the molecular weights of all multi-epitope vaccine constructs were in range from ~29 to ~40 kDa. The theoretical isoelectric point (PI) values of all constructs ranged from 5.46 to 8.71. The estimated half-life in in-vitro mammalian reticulocytes was 30 hours, >20 hours in in-vivo yeasts, and >10 hours in in-vivo E. coli. The instability index of Cholera toxin B-vaccine, PADRE-vaccine, and 50S Ribosomal protein L7/12 ranged from ~28 to ~35 which indicated these constructs were stable at various temperatures ⁴⁰. However, the instability index of RS09-vaccine and beta-defensin-vaccine was higher than 65, indicating their instability property ⁴⁰. Gravy scores of all vaccines ranged from -0.427 to -0.699, showing hydrophilic nature ^40,41. In line with Gravy scores, solubility scores calculated by SOLpro server were in range of ~0.72 to ~0.98, indicating high solubility ⁴². Aliphatic index scores of Cholera-vaccine, PADRE-vaccine, RS09-vaccine, and 50S Ribosomal protein L7/12 were in range of 65.88 to 77.81, representing high thermostability whereas the aliphatic index of beta-defensin-vaccine was 35.21, indicating low thermostability ⁴³. Finally, vaccine was also tested for the signal peptide sequences; however, there was no signal peptide sequence found within all tested vaccines. Finally, vaccine was also tested for the signal peptide sequences; however, there was no signal peptide sequence found within all tested vaccines. Collectively, based on the all above predictions, PADRE-vaccine demonstrated overall improvement in terms of antigenicity, solubility, and stability without compromised allergenicity and toxicity. Thus, PADRE-vaccine was recognized as the lead vaccine construct for immunological translation and chosen for further analysis.

Secondary structure prediction, three-dimensional Structure Predictions, refinement, and validation. PADRE-vaccine showed the overall improvement in both immunological properties and physicochemical properties; therefore, it was used to predict secondary structure and three-dimensional structure. The secondary structure of vaccine was predicted by using PSIPRED 4.0 server to identify the features. The predicted secondary structure resulted in 44.6% Coils, 33.7% α-Helix, and 21.8% β-Strands as shown in Fig. 3. Additionally, the three-dimensional structure of PADRE-vaccine was predicted using Alphafold 2. Five there-dimensional structure models were generated by Alphafold 2, and the model with the highest pLDDT score was selected for further analysis. To improve the accuracy of the predicted three-dimensional structure model, the lead model obtained from Alphafold 2 was further refined by GalaxyWEB server for the refinement, resulting five refined models with improved quality demonstrated by quality assessment parameters as shown in Table 4. Model 2 was selected based on improved Molprobity and Rama favored scores of 1.055 and 98.6, respectively, which indicated the high quality of the model. PyMol was used to visualize the three-dimensional model of the initial and refined three-dimensional structure models (Fig. 4).

Model 2 was further analyzed for validation with SAVES 6.0 server by ERRAT and PROCHECK. The selected model exhibited a high quality score with 86.9281% according to ERRAT. According to PROCHECK for evaluation of Ramachandran plot, there were 97.5% residues found in preferred region, 1.6% in allowed region, 0.4% in generously allowed regions and only 0.4% in forbidden regions as shown in Fig. 5a. Additionally, ProSa-Web server was used to compute the z-score of the model. The z-score calculated by ProSa-web server was -4.62 as shown in Fig. 5b. Collectively, the results from ERRAT, PROCHECK, and ProSa-web represented excellent quality of the predicted three-dimensional model.

Molecular docking of TLRs and selected vaccine construct. The ClusPro 2.0 server was used to predict the binding affinity between PADRE-vaccine and TLR4. The results, which included cluster size, center energy score, and lowest energy score. Out of thirty best fit docking models, the models with the largest clusters size model for the complex were prioritized for further analysis. This model showed the score of -1375.4 for both center energy score and lowest energy as shown in Table 4. The lead model obtained from the ClusPro 2.0 server was further refined via REFINEMENT tool on the HADDOCK 2.4 server. The refined model was analyzed for Gibb’s free energy(ΔG) and dissociation constant (K_D) as well as other aspects including HADDOCK score, Cluster size, RMSD from the overall lowest-energy structure, Van der Waals energy, electrostatic energy, desolvation energy, restraints violation energy, buried surface area, z-score as shown in the Table 4. The predicted model of vaccine-TLR4 complex was selected. Binding energy (ΔG) of this model was -18.7 kcal/mol and dissociation constant (K_D) was 2.00 x 10^-14 M. With Van der Waals energy, electrostatic energy, desolvation energy, restraints violation energy, and buried surface area, these parameters contributed HADDOCK score of -808.4 +/- 7.9 for vaccine-TLR4 complex. Additionally, the cluster size of the complex was 20. Root Mean Square Deviation (RMSD) from the overall lowest-energy structure was 0.7 +/- 0.4 Moreover, a z-score of 0 showed that this model was exactly on the average score within the cluster. Overall, these parameters indicated that the selected model was stable and energetically favorable interaction.

In silico immune stimulation. C-IMMSIM v10.1 server was used to predict the immunization of candidate vaccine. The results from stimulation using the candidate vaccine included B-cell, helper T-cell, cytotoxic T-cell, natural killer cell (NK), macrophage (MA), dendritic cell (DC), and certain cytokines. PADRE-vaccine could trigger IgM and IgG production since primary immunization and dramatically elevated the production of IgM, IgG1, and IgG2 after the secondary and tertiary response as shown in Fig. 6a and 6c. In line with the elevated antibody titer, total B-cell populations, and particularly B isotype IgM and memory B-cell were induced since the first injection (Fig. 6b). Those total B-cell, IgM, memory B-cell, and IgG1 populations were elevated dramatically, while B isotype IgG2 could also be induced after the second and third injections as shown in Fig. 6b. Noticeably, a decrease in the antigen was detected as shown in Fig. 5a, and these phenomena resulted from the generation of memory B-cell population (Fig 6b). Also, Fig. 6c indicated that plasma B lymphocyte (isotype: IgM+IgG) could be noticeably elevated. The active B-cell population per state increased significantly as well as became consistently high after the immunization as demonstrated by Fig. 6d. According to Fig. 6e, the total help T-cell population was increased significantly from first, second and third injections. Particularly, both memory and non-memory helper T-cell were dramatically elevated. Likewise, active, and resting helper T-cell population per state could be elevated significantly as indicated by Fig. 6f. Although resting cytotoxic T-cell population per state went down after the first immune response, it dramatically elevated over time after the tertiary immune response as shown in Fig. 6g. Total NK cell, total MA population per state, especially resting MA, and DC population per state (for both active and resting) were also generated by the immunization as demonstrated by Fig. 6H, 8I, and 8J. Regarding Fig. 6K, the titer of interferon-γ (IFN-g) was induced significantly by all three injections. The increased transforming growth factor-β (TGF- β), interleukin 10 (IL10) and interleukin 12(IL 12) were observed. The unelevated danger level D on the same figure indicated that risk was extremely low. This represented a favorable reaction to immunization.

Codon optimization and in silico cloning. Codon optimization was performed using Java Codon Adaptation Tool (JCAT) for protein expression in E. coli. The protein sequence of PADRE-vaccine was reverse-translated to DNA sequence. The DNA sequence was submitted to JCAT server for codon optimization. The Codon Adaptation Index (CAI) and GC content of the codon optimized sequence was 1.0 and 51.11% (Supplementary Dataset 2), respectively. These scores indicated the optimized nucleotide sequence was found within the range of optimum CAI (0.8 - 1.0) and GC Content (30 - 70). Finally, the optimized nucleotide sequence above was in-silico cloned by inserting into pET21a vector digested with NdeI and XhoI restriction enzymes using SnapGene 7.2.1, resulting a recombinant plasmid (pET-21a(+)-PADRE Mpox vaccine-6xHis tag) shown in Fig. 7.

NMA via iMODS server. The iMODS server and GROMACS software were utilized to perform molecular dynamic simulation (MD). iMODS was employed to analyze the vaccine-TLR complexes using normal mode analysis (NMA) to investigate the binding affinity of the vaccine-TLR4 complex and to investigate the stability and dynamic movements over time. The main-chain deformity was performed by measuring the capability of molecular deformation at each residue. High peaks represented the deformable regions of the vaccine-TLR complex, indicating flexible locations, while rigid regions were indicated by low values, as shown in Fig. 8. According to Fig. 8a, the vaccine-TLR4 complex demonstrated low distortion.

B-factor charts, shown in Fig 8b, indicated the average RMSD values of the vaccine-TLR complex by relating the mobility of computed complex in NMA and to the corresponding PDB field. The eigenvalues of the docked, illustrated in Fig. 8c, explained the motion stiffness. The required energy value for structural deformation was indicated by the eigenvalue, with the lower eigenvalue representing easier deformation. The eigenvalue of the interaction between vaccine and TLR4 6.239649 X 10^-7. According to Fig. 8d, the graphs of variance, which showed an inverse association with the eigenvalue, were shown. The purple color represented individual variance, while the green color represented cumulative variance for each normal mode of the vaccine-TLR complex. The covariance map of the vaccine-TLR complex illustrated coupling between two molecules of the complex (Fig. 8e). Red color represented correlated atomic motions, white color represented uncorrelated atomic motion, and blue color represented anticorrelated atomic motion in the regions of MD depicted in Fig. 8e. The elastic network map of vaccine-TLR4 complex shown in Fig. 8f depicted each gray dot as the formation of springs connecting corresponding pair of atoms. Flexible regions were represented by lighter dots, whereas stiffer regions were indicated by darker dots. Collectively, all these results highlighted the stable interaction between vaccine and TLR4.

MD analysis via GROMACS. To measure the stability of vaccine-TLR4 complex, the backbone atom RMSD was computed from initial point of time. According to Fig. 9a, the RMSD of the complex rapidly increased from the beginning to approximately 5 ns. After that, the structure stabilized until around 20 ns, with the range of approximately 0.6 to 0.5 nm. However, there was an increase after 20 ns until it became substantially stabilized after approximately 24 ns with the range of around 0.6 - 0.8 nm throughout the remaining simulation time. Additionally, RMSF was analyzed to assess the flexibility of vaccine-TLR complex. The high fluctuation of RMSF indicated highly flexible regions of the complex. The peak of fluctuation was 2.1589 nm of residue 636, as shown in Fig. 9b. Several highly fluctuation regions were identified, including the region between residue 409 – 519 (with the range of 0.8203 to 1.287nm), the region between residues 996 - 1222 (with the range of 0.7031 -1.2779 nm), the region between residue 628 – 653 (with the range of 1.0358 – 2.1589 nm), and the region between residue 1267 – 1558 with a very wide range of fluctuations between 0.2887 – 1.5996 nm. The last region contained several high and highest fluctuations residues, such as residue 1267 with 1.5996 nm, residue 1414, with 1.4612 nm, and residue 1558 with 1.5258 nm.

Rg was analyzed to determine the structural compactness of vaccine-TLR complex. As illustrated in Fig. 9c, the overall Rg was substantially stable throughout the simulation time. Furthermore, the stability of the complex could be measured with the number of hydrogen bonds. According to Fig. 9d, the result of the MD analysis illustrated the stability of the number of hydrogen bonds with a very narrow range between 10 and 11 throughout the simulation time.

Finally, the SASA could indicate the surface area of a vaccine-TLR complex exposed to solvent molecules. The results could imply the flexibility and amenability of protein-protein interaction. Illustrated by Fig. 9e, there was a fluctuation from the beginning of simulation time to around 20 ns, with the range of ~760 to ~720 nM². After 20 ns until 34 ns, SASA became more stable. Then, there was a sudden drop at around 36 ns; however, SASA maintained it stability during ~36 to 42 ns. Eventually, it increased again at simulation time ~43 ns, after that SASA became stable until the end of simulation time.

Monkeypox is a zoonotic double-stranded DNA virus in the family of orthopoxviral genus. Recently, there has been a global outbreak of monkeypox disease with 97,962 infected cases and 184 deaths according to the data from CDC as of June 30^th, 2024. To address the global outbreak, several smallpox vaccines have been introduced to offer cross-protection against monkeypox including ACAM2000, JYNNEOS (MVA-BN), and LC16 ⁴⁴. This also means non-specific vaccines are being used to provide protection against monkeypox. A small study using previous data from Africa found the smallpox vaccine might offer approximately 85% protection against monkeypox ⁴⁵.

The Advisory Committee on Immunization Practices (ACIP) recommends the ACAM2000 live-attenuated vaccinia virus vaccine for specific personnel groups. These groups include laboratory personnel routinely handling human infectious orthopox viruses (monkeypox, cowpox, variola) and medical personnel caring for vaccinia virus-infected patients ⁴⁶. However, ACAM2000 carries potential safety risks. Studies have reported occurrences of post-vaccinal encephalitis, eczema vaccinatum, and progressive vaccinia following vaccination ⁴⁶. Additionally, the presence of live vaccinia virus raises concerns about secondary transmission to both the vaccinated individual and those in close contact, and the potential development of myocarditis or pericarditis in some recipients ⁴⁷. Meanwhile, administration of the JYNNEOS vaccine has been associated with various side effects, including induration, chills, headache, sore throat, nausea, redness, myalgia, firmness/tightening, and pain ⁴⁴. A retrospective study conducted in a Northwestern US population cohort (Oregon) identified 10 cases of cardiac events following JYNNEOS vaccination between July and October 2022 ⁴⁸. Similar to JYNNEOS, LC16 is a live-attenuated, non-replicating vaccine approved for monkeypox prevention in Japan in August 2022 ⁴⁷. Most adverse events were reported as mild to moderate localized or systemic in nature. Notably, clinical trials and cohort studies have not identified serious adverse events such as encephalitis or symptomatic myocarditis ⁴⁴. While pre-clinical data suggests potential safety for immunocompromised and atopic dermatitis patients, widespread vaccination in these populations is not currently recommended due to insufficient clinical data ⁴⁴. The observed limitations of live-attenuated vaccines suggest the need to explore alternative vaccination strategies that offer comparable efficacy with reduced adverse effects.

To address the shortcomings, a multi-epitope vaccine was proposed as a candidate for this study because this novel type of vaccine exhibits a reduced capacity for eliciting pathological adverse effects due to the absence of unwanted components, compared to traditional vaccines ⁴⁹. Those unwanted components could be screened by using immunoinformatic tools. In tests with animal models and early clinical trial, multi-epitope vaccines induced cellular and humoral immune responses as well as elicited robust immune responses of the individual epitope against tumor, microbial, and viral infection ^49,50. In this study, epitopes were recruited from the monkeypox viral proteins as sequences of both structural and non-structural viral proteins were screened for only strong binding CTL, HTL, and LBL epitopes. Only strong binding CTL epitopes that could induce class I immunogenicity and strong binding HTL that could induce interferon-γ were recruited for the vaccine construct. Meanwhile, the highest antigenic LBL epitopes were recruited as well. These epitopes were also screened for allergenicity, toxicity, transmembrane regions, and human analogous proteome. From 200 monkeypox viral proteins, epitopes derived from 8 proteins including A40L, B6R, B9R, C2L, E4R, F8L, H3L, and J3R/J1L. These proteins were found to be able to potentially induce adaptive immune system and cytokines. Particularly, previous studies found B6R and H3L to be immunodominant as they provided CTL, HTL, LBL, and interferon-γ stimulation ^26–30. Vaccinia viral proteins B5R and H3L, homologous proteins of monkeypox viral protein B6R and H3L, respectively⁵¹, were found to be immunodominant ^32,33,52,53. F8L was another highly dominant immunogenic protein that was found to trigger both HTL and CTL ^54,55. This protein was also tested in vivo and found that it could induce CTL in non-human primates ⁵⁵. E4R was another monkeypox viral protein that was reported to induce antibodies in vivo among prairie dogs ³¹. Meanwhile, monkeypox viral protein A40L and C2L were not directly mentioned in the previous studies. However, vaccinia viral proteins A38L and K2L were homologous proteins to A40L and C2L, respectively ⁵¹. They were found to be crucial ligands to MHC class II and HTL ^32,33. B9R is homologous to vaccinia viral protein B8R ⁵¹. This protein was found to be immunodominant in CTL stimulation ^56,57. Finally, J3R/J1L which is a chemokine binding protein, plays the critical role in modulating and preventing apoptosis in host cell to facilitate viral infection ³⁴. The homologous vaccinia viral protein B29R of J3R/J1L was reported to involve acute and memory CTL stimulation with recombinant antigen ⁵⁸.

Those selected epitopes from proteins above were investigated for the epitope conservancy. According to the previous studies on vaccine design, there was still no vaccine that could provide board-spectrum protection against all sub-clades. Even though the most virulent clade II with less deadly effects caused the global outbreak in 2022 with the sub-clade IIb ^59–61, High fatal clade I was observed for the outbreak again during 2023-2024 ^59,61. Therefore, this study aimed to provide the board-spectrum protection from both high lethal and high virulent clades because clade I strains are generally determined as the deadliest clade with less virulence, whereas clade II strains are highly virulent, but less lethal ^59–61. Accordingly, the completed genome of Clade I Zaire-96-l-16 and 7 variants of Clade II including Clade IIa USA 2003, Clade llb-A Nigeria 2018, Clade IIb-A.2 USA 2022, Clade IIb-A.1 UK 2018 (UK P2), Clade IIb-A.1.1 USA 2021, Clade IIb-B.1 France 2022 were employed to the IEDB Epitope Conservancy Tool. Almost all epitopes selected for the vaccine construct showed 100% conservancy. Only one HTL epitope, SMVFEYRASTVIKGP presented a single amino acid substituted from valine to isoleucine. A valine in Clade I strains was substituted by isoleucine in Clade IIa, Clade llb-A, Clade IIb-A.2, Clade IIb-A.1, Clade IIb-A.1.1, and Clade IIb-B.1. However, negligible effect could be contributed by valine to isoleucine substitution because both valine and isoleucine are categorized in hydrophobic amino acid group.

The vaccine construct model from selected epitopes was improved further with adjuvants and linkers. Studies have shown that linkers incorporated into vaccine constructs can enhance their stability ^62,63. These linkers are thought to function by creating spatial separation between individual epitopes, thereby minimizing potential steric hindrance and ensuring the independent immunogenicity of each epitope upon vaccination ⁶². In the meantime, adjuvant was also fused to the vaccine model to improve immune response. Moreover, adjuvants can exert their immunostimulatory effects through mechanisms including, Antigen depot promoting prolonged exposure to the immune system, Activation of innate immunity via pathogen recognition receptors (PRR) engagement, immune cells co-stimulation, immunomodulation, particularly antigen-presenting cells (APCs) maturation ^64,65. The results of physicochemical properties in this study could indicate the improvement of the vaccine model, especially the best selected model with PADRE adjuvants. Compared to other designed vaccine models, PADRE adjuvant vaccine had the highest antigenicity, high solubility, and improved stability without being potentially allergenic and toxic.

After that, PADRE adjuvant vaccine model was further performed molecular docking and MD with TLR4 because this TLR4 plays a crucial role in antiviral infection ^66,67. TLR4 signaling complex might physically and hydrophobically interact with the hydrophobic pocket of a co-receptor called MD-2 and hydrophobic fusion peptides on the viral proteins ⁶⁶. Another potential factor is glycosylation, as all TLR4-activating viral proteins have glycosylation ⁶⁶. Moreover, the way viral proteins assemble into units (oligomeric state) might also play a role in the interaction with the TLR4 signaling complex. ⁶⁶. Therefore, the TLR4 pdb file was retrieved from the Protein Data Bank (PDB ID: 4G8A); however, some missing residues were indicated by pdb file information. Consequently, Modeller10.4 ^68,69 and EMBOSS NEEDLE ⁷⁰ were used to perform pdb file improvements before docking and MD.

The results of molecular docking with the ClusPro 2.0 server ⁷¹ score demonstrated a favorable score of -1375.4 for both center energy score and lowest energy. Similarly, binding energy and dissociation constant performed by the HADDOCK 2.4 server ^72–74 were favorable with binding energy (ΔG) of -18.7 kcal/mol and K_D of 2.00 x 10^-14 M. Nevertheless, there are limitations of these docking servers. With rigid-body estimation, only moderate molecular conformations were allowed upon binding ⁷⁵. In fact, an important aspect of correct approximating the binding geometries is flexibility ⁷⁶ Additionally, a reliable approximation of key thermodynamic observations (such as the binding free energy) is not allowed ⁷⁶. Furthermore, only a rigid picture of the binding process is produced, leading to the lack of kinetic quantities approximation ⁷⁶. To address the limitations, the iMOD server ⁷⁷ was used to analyze the rigidity and deformability which indicated the stability of the vaccine-TLR4 complex. In addition, GROMACS software ^78,79 was employed to analyze the stability and dynamics of the complex structure within a timeframe of 50 ns and under more realistic conditions of solvation, neutralization (with Na and Cl ions), 310 K temperature and 1atm pressure. At the meantime, immunization simulation performed with C-ImmSim server ^80–82 supported the idea that the PADRE vaccine had the potentials to bind effectively with TLR4 because results of immunization indicated both innate and adaptive immune response.

Eventually, in vitro and in vivo studies are indispensable for validating this broad-spectrum multi-epitope vaccine design. Critical steps include protein expression in E. coli to confirm its expressibility as predicted by physicochemical properties, as well as in vivo studies in model organisms. Furthermore, the potential of this vaccine design can be significantly enhanced by exploring advanced delivery systems such as mRNA and outer-membrane vesicles (OMVs). These additional studies will not only affirm the efficacy and safety of the vaccine but also open new avenues for innovative approaches in vaccine development, offering a robust defense against diverse and evolving viral threats.

Retrieval of monkeypox viral proteins. The protein sequences of the monkeypox virus Clade I (Congo/Central Africa) were retrieved from the GenBank database with the accession number DQ011154.1. This clade has the highest lethality and causes severe symptoms compared to other virulent clades. ^59,60. Although clade IIb caused the global pandemic in 2022, it had less mortality rate than clade I ^59,60. More importantly, infected patients with clade I are recently observed again in Democratic Republic of the Congo during 2023 – 2024 ^59,61,83. According to the lethal rate and reemerging cases of Clade I, MPXV strain Congo_2003_358 was utilized as a benchmark for collection of MPX proteins.

Out of 200 protein candidates, 24 proteins, including structural and non-structural proteins retrieved from the complete genome were selected based on antigenicity and allergenicity, and toxicity. VaxiJen v2.0 ^84–86 server (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) was used to screen for antigenic viral proteins. The accuracy of the VaxiJen in protective antigen projection of the viral protein ranges from 70 to 89% ⁸⁵. Additionally, AllergenFP v1.0 (https://ddg-pharmfac.net/AllergenFP/) was used to predict allergenicity of proteins ⁸⁷. Only proteins with an antigenic probability score greater than 0.4 and non-allergic property were further utilized for epitope prediction.

Prediction of cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and Linear B-lymphocyte (LBL).The cytotoxic T-lymphocyte (CTL) epitopes of the selected MPXV proteins were predicted by using both NetMHCpan 4.1 BA and NetMHCpan 4.1 EL (https://services.healthtech.dtu.dk/services/NetMHCpan-4.1/) as a prediction method ⁸⁸. The HLA alleles including HLA-A0101, HLA A0201, HLA-A0301, HLA-A2402, HLA-A2601, HLA-B0702, HLA-B0801, HLA-B2705, HLA-B3901, HLA-B4001, and HLA-B5801 were selected to screen for CTL epitopes. Since the vast majority of peptides presented by MHC class I are 9 amino acids, the peptide length was set at 9 amino acids. Additionally, only CTL epitopes demonstrating strong binding results were selected. The predicted epitopes with strong binding were further investigated an ability to trigger immunogenicity via Class-I Immunogenicity tool in IEDB (http://tools.iedb.org/immunogenicity/) ⁸⁹ before the further assessment.

Similarly, the helper T-lymphocyte (HTL) epitopes of each viral protein were predicted by NetMHCpanII 4.1 BA and NetMHCpanII 4.1 EL (https://services.healthtech.dtu.dk/services/NetMHCIIpan-4.1/) ⁹⁰. DRB1_0101, DRB1_0301, DRB1_0401, DRB1_0701, DRB1_0801, DRB1_0901, DRB1_1001, DRB1_1101, DRB1_1201, DRB1_1301, DRB1_1501, and DRB1_1602 were selected as targeted HLA Alleles to screen for HTL epitopes. The length of the epitopes was set as default at 15 amino acids. Only the epitopes with strong binding results were selected. Then, interferon-γ activation potential of the HTL epitopes was predicted by IFNepitope (http://crdd.osdd.net/raghava/ifnepitope/predict.php) ⁹¹

Furthermore, the linear B-lymphocytes (LBL) epitopes of monkeypox proteins were predicted using the Bepipred Linear Epitope Prediction 2.0 server (https://services.healthtech.dtu.dk/services/BepiPred-2.0/) with the default threshold of 0.5 ⁹². All these predicted epitopes (CTL, HTL, and LBL) were validated for allergenicity, antigenicity, and toxicity screening.

Assessment of CTL, HTL, B-cell epitopes on allergenicity, antigenicity, and toxicity parameters.The allergenicity of all selected epitopes of HTL, CTL and LBL was determined using AllergenFP v1.0 ⁸⁷. The Vaxijen v2.0 server was used to evaluate antigenicity with a threshold of 0.4 ^84–86. The ToxinPred2 server (https://webs.iiitd.edu.in/raghava/toxinpred2/) was used for toxicity prediction, with an SVM (Swiss-Prot) based prediction method ⁹³. Only peptides demonstrating non-allergenic, antigenic, and non-toxic properties were selected for analysis of epitope conservancy.

Screening of transmembrane region. Before vaccine construction, candidate epitopes were predicted for transmembrane regions. The transmembrane regions of all selected epitopes were predicted by DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) ⁹⁴. The predicted transmembrane segments were omitted from vaccine construction to avoid low-yielding and unstable expression of protein due to the hydrophobic effect ^35–37.

Epitope conservancy for the broad-spectrum coverage of vaccine over different clades. To generate a broad-spectrum vaccine against various clades of the monkeypox virus, all selected CTL, HTL, and LBL epitopes were investigated for epitope conservancy across seven monkeypox virus clades, including Clade I (Zaire-96-l-16), Clade IIa (USA 2003), Clade llb-A (Nigeria 2018), Clade IIb-A.2 (USA 2022), Clade IIb-A.1 (UK 2018 P2), Clade IIb-A.1.1 (USA 2021), Clade IIb-B.1 (France 2022), using the IEDB Epitope Conservancy Tool (http://tools.iedb.org/conservancy/) ⁹⁵. Only highly conserved epitopes demonstrating percent identity higher than 90% were retrieved for vaccine construction.

Autoimmune screening by homology analysis of epitopes with human proteomes. To avoid the possibility of inducing autoimmune diseases or cross-reactivity in the host, all selected CTL, HTL, and LBL epitopes obtained in section 2.5 were blasted against the Homo sapiens proteome (Taxonomy ID: 9606) via the Peptide Search server (https://www.uniprot.org/peptide-search) ⁹⁶. Only epitopes exhibiting non-homologous identity to the human proteome were subsequently selected for vaccine construction.

Population coverage. To quantify the global impact of the vaccine, the IEDB population coverage tool (http://tools.iedb.org/tools/population/iedb_input) was employed to analyze the worldwide distribution of HLA alleles targeted by our selected MHC class I and II epitopes ⁹⁷. The HLA allele genotypic frequencies from the Allele Frequency database (http://www.allelefrequencies.net/) were used to provide the information on genetic variations in human populations ⁹⁸. This database offers data for over 115 countries and categorizes 21 ethnicities within 16 geographic regions ⁹⁸. It also allows users to define custom populations with specific allele frequencies. The program calculates population coverage for various groups and generates an average value.

Construction of the multi-epitope subunit vaccine. The multi-epitope vaccines were constructed by combining the final epitopes of CTL, HTL, and LBL with separate linkers (AAY, HEYGAEALERAG, GPGPG, and KK). To enhance immunogenicity and render the longevity of innate and adaptive immunity ^38,39, a peptide adjuvant was fused to the multi-epitope vaccine. Five different peptide adjuvants used in this study included PADRE, beta-defensin 3, 50S ribosomal protein L7/12, RS-09, and cholera toxin B subunit (CTB). Each adjuvant was N-terminally fused to multi-epitope vaccine, thereby yielding six multi-epitope vaccine constructs.

Physicochemical, antigenicity, allergenicity, toxicity, and solubility of the vaccine. The physiochemical properties of each multi-epitopes vaccine construct, including molecular weight, grand average of hydropathicity (GRAVY), theoretical isoelectric point (pI), instability index, and half-life, were evaluated using the ProtParam tool of the ExPASy database server (https://web.expasy.org/protparam/) ⁴⁰. The antigenicity and toxicity of the designed vaccines were determined using AllergenFP v. 1.0 (http://ddg-pharmfac.net/AllergenFP/) ⁸⁷ and the ToxinPred2 server (https://webs.iiitd.edu.in/raghava/toxinpred2/) ⁹³, respectively. Allergenicity was identified by VaxiJen v2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) ^84–86. The SOLpro server (http://scratch.proteomics.ics.uci.edu/) was used to predict the solubility of the designed vaccines by SVM-based method to predict the solubility of vaccine sequence ⁴². Additionally, SignalP6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/) was employed to identify signal peptides regions of the recombinant proteins ⁹⁹.

Secondary prediction, tertiary Structures Predictions, refinement, and validation. The secondary structure features (α-helixes, β-sheets, and random coils) of selected vaccine construct were predicted using PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) ¹⁰⁰. The tertiary structure of the selected vaccine was predicted using AlphaFold2 by implementing the fast homology search of MMseqs2 with AlphaFold2 ¹⁰¹. Following the initial three-dimensional (3D) modeling, the primary 3D models with the highest score were further refined by submission to the GalaxyRefine module on the GalaxyWEB server (https://galaxy.seoklab.org/) ^102,103. This tool optimizes unreliable loops and terminal regions through an optimization-based refinement method and generates five refined models from each initial structure ¹⁰⁴. The model with the lowest MolProbity score was selected as the most well-defined for each vaccine construct. These chosen models were further evaluated for quality validation using ERRAT and PROCHECK from SAVES v6.0 (https://saves.mbi.ucla.edu/), and ProSA-Web servers (https://prosa.services.came.sbg.ac.at/prosa.php).

The ERRAT program evaluated overall structural integrity by quantifying non-bonded interactions between neighboring atoms ¹⁰⁵. Models with an overall quality factor exceeding 85 were considered reliable. The Ramachandran plot generated by PROCHECK tool evaluated the stereochemical correctness of the protein backbone, with more residues located in favored regions indicating increased model accuracy ^106–109. Lastly, the ProSA-Web server, through its Z-score prediction, provided an overall quality assessment in which positive values suggested potential structural issues ^110,111.

Molecular docking of Toll-like receptor 4 (TLR4) and the lead vaccine construct.

To determine whether the lead vaccine can trigger an immune response via TLR4, molecular docking evaluation between the lead vaccine and TLR4 (PDB ID: 4G8A) were performed to predict the formation of a persistent interaction between the complexes ¹¹².

To bridge gaps caused by some missing residues in the retrieved PDB file of TLR4, MODELLER10.4 was deployed to complete missing amino acid residues prior to the molecular docking analysis to obtain the most accurate outcomes ^68,69. After improving PDB file, the ClusPro 2.0 server (https://cluspro.org/login.php) was utilized to measure the binding energy ⁷¹ between the lead multi-epitope vaccine and the TLR4 receptor. Thirty model structures were generated for the docking scenario, and ten models demonstrating largest clusters were prioritized for further analysis.

To enhance structural precision, the top-ranked model obtained from the ClusPro 2.0 server was further refined using the HADDOCK 2.4 server (https://wenmr.science.uu.nl/haddock2.4/) ^72,113. This server optimizes complex structures by strategically rearranging side chains within restricted interfaces and employing soft rigid-body refinement techniques^72,113. Subsequently, the refined docked model was assessed for binding energy using the PRODIGY server (https://bianca.science.uu.nl/prodigy/), providing quantitative insights into its binding affinities ^72–74. PyMOL was used to visualize structural figures.

In silico immune stimulation. The selected vaccine was analyzed via the C-IMMSIM tool (https://kraken.iac.rm.cnr.it/C-IMMSIM/) to predict the immune efficacy of our designed vaccine ^80–82. The C-IMMSIM server can identify epitopes through Position Specific Scoring Matrix (PSSM) approaches to simulate immune interactions ^80–82. For the immune simulation, the simulation time steps were set to 1050 where 1-time step equals 8 h. The time step of each injection point was set at 1, 84, and 168, respectively. The dose of each antigen injection was set at 1,000 and the time interval between each injection was four weeks, which is the recommended interval for vaccination.

Codon optimization and in silico cloning. The vaccine construct was performed the codon optimization by using Java Codon Adaptation Tool (JCAT) (http://jcat.de/) ¹¹⁴ with E. coli strain K12 as the organism. After codon optimization, the vaccine sequence was inserted into the expression vector pET21a (+), digested with NdeI and XhoI, using Snap Gene 7.2.1.

Molecular dynamics (MD) simulation. The best docking model obtained from molecular docking analysis was analyzed by iMODS server to predict the stability of the vaccine-TLR complex based on Normal Mode Analysis (NMA) to predict the rigidity and deformability⁷⁷. The iMODs server provides information on deformability graph, B-factor graph (RMSD values), Eigenvalues, variance plot, covariance map, and elastic network map. Additionally, the structural dynamics and stability of the docked vaccine-TLR complex were analyzed by using GROMACS v2024 software ^78,79. CHARMM36 forcefield ^115,116 was adopted to create the topology file for MD analysis. TIP3P water model ^116–118 was also utilized for solvation of vaccine-TLR complex. Afterwards, Na and Cl ions were added to neutralize the system, followed by performing system energy minimization. NVT of each system was performed at 310 K, and the modified Berendsen thermostat ¹¹⁹ was employed to perform temperature equilibration. Parrinello–Rahman barostat ¹²⁰ was also employed. Pressure was adjusted at 1 atm for NPT simulation with a time constant of 2 ps. Then, the leapfrog algorithm was used to integrate the equations of motion with a time step of 2.0 fs.

The vaccine-TLR complex system was run for a 50 ns simulation under constant temperature and pressure. LINCS algorithm ¹²¹ was used for the constraint of hydrogen bonds. The Particle mesh Ewald (PME) method ¹²² was used to analyze long-range electrostatic energy. Additionally, short-range Coulomb electrostatic and Van der Waals cutoffs of 1.0 nm were used to perform MD. The analysis results of the MD analysis provided by the GROMACS software include root mean square deviation (RMSD), root mean square fluctuations (RMSF), radius of gyration (Rg), hydrogen bonds, and total solvent accessible surface area (SASA) of the system.

Author contributions. J.P. designed all research, performed all research, analyzed data, and wrote the paper. P.L. and P.J. performed three-dimensional structure prediction, structural refinement, in silico cloning, and data analysis. T.S. predicted physicochemical properties. B.M. conceptualized the project, designed research, analyzed data, and wrote the paper.

Acknowledgements. We thank Dr. Charnon Pattiyanon, Jarukit Suchat, Yan Trostin from CMKL University for kindly providing Apex server used in molecular dynamics simulation in this research.

Additional Information - competing financial interests. The authors declare no competing financial interests.

Data availability statement

All data generated or analyzed in this study are provided within the manuscript and supplementary information files.

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Engineering a broad-spectrum multi-epitope vaccine to combat emerging monkeypox virus by immunoinformatic approaches

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