Accelerating Cancer Vaccine Development for Human T-Lymphotropic Virus (HTLV) Using a High-Throughput Molecular Dynamics Approach

Human T-lymphotropic virus (HTLV), a retrovirus belonging to the oncovirus family, has long been linked to be associated with various inflammatory and immunosuppressive disorders. To combat the devastating impact of this virus, our study employed a reverse vaccinology approach to design a multi-epitope-based vaccine targeting the highly virulent subtypes of HTLV. We conducted a comprehensive analysis of the molecular interactions between the vaccine and Toll-like receptors (TLRs), providing valuable insights for future research on preventing and managing HTLV-related diseases and any possible outbreaks. The vaccine was designed by focusing on the envelope glycoprotein gp62, a crucial protein involved in the infectious process and immune mechanisms of HTLV inside the human body. Epitope mapping identified T cell and B cell epitopes with low binding energies, ensuring their immunogenicity and safety. Linkers and adjuvants were incorporated to enhance the vaccine’s stability, antigenicity, and immunogenicity. Two vaccine constructs were developed, both exhibiting high antigenicity and conferring safety. Vaccine construct 2 demonstrated expected solubility and structural stability after disulfide engineering. Molecular docking analyses revealed strong binding affinity between the vaccine construct 2 and both TLR2 and TLR4. Molecular dynamics simulations indicated that the TLR2-vaccine complex displayed enhanced stability, compactness, and consistent hydrogen bond formation, suggesting a favorable affinity. Contact analysis, Gibbs free energy landscapes, and DCC analysis further supported the stability of the TLR2-vaccine complex, while DSSP analysis confirmed stable secondary structures. MM-PBSA analysis revealed a more favorable binding affinity of the TLR4-vaccine complex, primarily due to lower electrostatic energy. In conclusion, our study successfully designed a multi-epitope-based vaccine targeting HTLV subtypes and provided valuable insights into the molecular interactions between the vaccine and TLRs. These findings should contribute to the development of effective preventive and treatment approaches against HTLV-related diseases.

arguably been associated to the development of HAM/TSP in infected patients like HTLV1 (10). 83 Moreover, HTLV-2 was also found to be associated with the onset of neuropathic disorders, 84 including meningitis and chronic inflammatory demyelinating polyneuropathy (CIDP) and 85 affecting patients clinical outcome negatively (11,12). The global impact of HTLV-1 and HTLV-86 2 infections is alarming, with an estimated 5-10 million people infected worldwide (13). HTLV-1 87 is highly prevalent in indigenous populations in Japan and South America, while HTLV-2 is 88 primarily found in Central and South America (14).  However, developing a vaccine against HTLVs has been challenging due to the ability of the virus 104 to integrate its genetic material into the host genome, making it difficult to target with a vaccine. 105 Additionally, the presence of a few distinct viral proteins that interfere with the immune system 106 and the variability of the virus further complicate vaccine development (11,19). Hence, 107 developing a vaccine targeting the effective virulent protein against HTLVs is crucial due to their 108 intricate mechanism of infection and evading the host defense system. Study suggests that 109 candidates able to boost anti-HTLV-envelope-glycoprotein antibodies may have potential roles in 110 combating HTLV infection (20). 111 This study focused on targeting the Envelope Glycoprotein GP62 of the viruses as a potential 112 antigen for the subunit vaccine. Glycoproteins, located on the outer layer of the viral envelope, are 113 easily recognized by the immune system. Enveloped glycoprotein which is a surface protein (SU) 114 of HTLV attaches the virus to the host cell by binding to its receptor, while the transmembrane 115 protein (TM) undergoes a conformational change upon this interaction with the host cell, activating 116 its fusogenic potential and leading to membrane fusion at the host cell plasma membrane. This 117 fusion process allows the viral nucleocapsid to enter the cytoplasm of the host cell (21,22). 118 Finally, the aim of the study is to design an effective polyvalent subunit vaccine against the major 119 causative agents of HTLV-related diseases i.e., HTLV-1, HTLV-2 using high-throughput 120 bioinformatics strategies. Additionally, given that the pathogenic mechanism and virulence of 121 other subtypes i.e., HTLV-3 remain under investigation, we also incorporated HTLV-3 subtype in 122 our experimental design. However, since the virulent protein sequence of HTLV-4 is yet not 123 reported in public repository, we didn't consider HTLV-4 in our study. Despite the challenges in

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The stepwise methodology of the entire study is depicted in Supplementary Figure S1  PDB database, and the refined 3D structure of the multi-epitope construct served as the ligand.

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The binding affinity between the vaccine construct and TLR2 and TLR4 was calculated using the 196 ClusPro 2.0 server and H-dock server (https://cluspro.bu.edu/login.php) (42). The best-docked 197 complex was identified based on the lowest energy-weighted score and docking efficiency.

Strain and protein selection with biophysical property analyses 267
The protein sequences of the target protein (Envelope glycoprotein gp62) in FASTA format were 268 retrieved from the UniProt database. constructive representations of vaccine construct-1 and 2, respectively, and the conservancy of the 286 epitopes among the selected viral strains is shown in Figure 2.  can be found in Table 2 and Supplementary Table S8, respectively. Based on the assigned 316 docking score, solubility, and other desired criteria, vaccine construct-2 was selected for additional 317 Molecular Dynamics (MD) simulations using the Gromacs 2020.4 package to further evaluate its 318 interaction and binding affinity with TLR2 and TLR4 (Berendsen, 1995). The RMSDs in the C-α atom were analyzed independently for each chain of TLRs and vaccine 324 chain. In the TLR2-vaccine complex, chain D has a higher number of fluctuations throughout the 325 simulation period with an average of 0.390 nm ( Figure 5A, Table 3), however reaching beyond 326 0.5 nm occasionally. Chain A has a slightly higher RMSD after around 50 ns than chains B and C.

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The average RMSD for chain A is 0.351 nm, slightly higher than the average RMSD of 0.314 nm     bond with different residues of chain C viz. Asn294, Ser298, Phe325, and chain D residues viz.

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Asn294, Ile304, Ser329, Asn297, and Asp299 ( Figure 8B). The trajectory at 50 ns showed that 397 the hydrogen bond between the residues Gly1, Asn4, Thr35, and Lys45 from vaccine chain and 398 chain C residue Glu264, and chain D residues Asn294, Ile304, and Asp299 remained stable with 399 no new hydrogen bonds ( Figure 8C). The trajectories captured at 75 ns showed that the residues 400 Thr10, Lys32, and Lys45 from the vaccine chain form a hydrogen bond with chain C residue 401 Asn294, Ser298, and Asp299 and chain D residue Asp299 ( Figure 8D). The trajectory at 100 ns 402 showed these hydrogen bonds remained intact and additionally a new hydrogen bonds between 403 residues Gly1, Lys8, Thr9, Arg14, and Arg42 from vaccine chain and chain C residues Asp327, 404 Gly291, Val292, Gly293 and chain D residue Glu336 ( Figure 8E). 405 In the case of the TLR4-vaccine complex, the initial equilibrated trajectory showed many hydrogen 406 bonds between the vaccine chain and TLR4 B and D chains. The residues Arg12, Arg14, Arg17,

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Cys18, Ser22, Lys26, Cys33, Ser34, Arg36, Arg38, Arg43, Lys45, and Thr35 formed a hydrogen 408 bond with chain B residues Arg355, Arg382, Glu425, Glu474, Asp502, Asp428, Glu603, Tyr403, residue-residue contacts between these TLR chains and the vaccine chain was analyzed through 422 contact map analysis (Figure 10). 423 The contact maps results show that the TLR2 chain C has slightly a greater number of residue-424 residue contacts with the vaccine chain compared to residue-residue contacts between chain D and 425 the vaccine chain. More residue-residue contacts were found between TLR4 chain B, and the 426 vaccine chain compared to residue-residue contacts between TLR4 chain D and the vaccine chain 427 (Figure 10A-B). The secondary structural changes which occur during MD simulation were analyzed from DSSP 449 analysis. Especially, the TLR chains which are in close contact with vaccine chains were analyzed.

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The results showed that the TLR2 chain C and chain D are relatively stable for secondary structural 451 changes ( Figure 11C-D). However, the vaccine chain showed major structural changes in the loop   Standard deviations are given in parentheses.

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Two vaccines were developed using the identified epitopes, and both were found to be highly 504 antigenic, non-allergenic, and non-toxic, confirming their safety and potential to trigger a sufficient 505 immune response. Vaccine construct 2 was found to be soluble while vaccine construct 1 was binding affinity compared to the vaccine construct bound to TLR4. The major difference found in 591 the energetic is in electrostatic energy in TLR4 which is significantly lower than TLR2.

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Overall, molecular dynamics simulations showed enhanced stability and favorable affinity in the 593 TLR2-vaccine complex, supported by contact analysis and secondary structure stability, while 594 MM-PBSA analysis favored the TLR4-vaccine complex due to lower electrostatic energy.