Immunoinformatics Design of Multi-Epitope Peptide-Based Vaccine Against Cyprinid Herpesvirus-3 (CyHV-3) Targeting Thymidine Kinase Proteins

Mohammad Habibur Rahman Molla^1,2*, Mohammed Othman Aljahdali², Amer H Asseri³, Bushra Jahan⁴, Md Afsar Ahmed Sumon⁵, Nazmul Ahasan Maruf⁶, Khalid Suliman Alsohibany⁷, M Aminur Rahman⁸, Earl T Larson⁹, Nushrat Jahan Maliha¹⁰, Mohammed Moulay¹¹, Christopher L Brown^1,2*
1Rubenstein School of Environment and Natural Resources, University of Vermont, Burlington, VT 05405, USA
²Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21598, Saudi Arabia
³Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia
⁴Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna, Bangladesh
⁵Marine Biology Department, Faculty of Marine Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
⁶Department of Computer Science, Faculty of Computing and Information, King Abdulaziz University, Jeddah-21589, Saudi Arabia
⁷Ministry of Health, Riyadh 93160, Saudi Arabia
⁸Department of Fisheries and Marine Bioscience, Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore 7408, Bangladesh
⁹Department of Biological Sciences, St. Johns River State College, Orange Park, FL, USA
¹⁰Department of Animal Husbandry, Patuakhali Science and Technology University, Dumki-Patuakhali Highway 8602, Bangladesh
¹¹Embryonic Stem Cell Research Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia
¹²FAO World Fisheries University Pilot Programme, Pukyong National University, Busan 47340, South Korea; Current Postal Address: 1212 E. Schwartz Blvd., Lady Lake, Florida USA 32159
*Correspondence author: MHR Molla, Rubenstein School of Environment and Natural Resources, University of Vermont, Burlington, VT 05405, USA and Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21598, Saudi Arabia and CL Brown, FAO World Fisheries University Pilot Programme, Pukyong National University, Busan 47340, South Korea; Current Postal Address: 1212 E. Schwartz Blvd., Lady Lake, Florida USA 32159; Email: [email protected]; [email protected]

Published Date: 05-04-2024

Copyright^© 2024 by Molla MHR, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The common carp Cyprinus carpio is a freshwater teleost and is among the most economically significant fishes in aquaculture throughout the world. Taxonomically, C. carpio are a complex of species including subspecies Cyprinus carpio carpio. C. carpio are now threatened by Cyprinid Herpesvirus-3 (CyHV-3), the causative agent of Koi Herpesvirus Disease (KHVD), which causes severe morbidity and mortality in ornamental koi and common carp and can infect or be transmitted by other species. Despite these devastating circumstances, effective vaccinations or other medications for the control of KHVD are not readily available. For this reason, the aim of the current study was to formulate a multi-epitope vaccine against Cyprinid Herpesvirus-3 (CyHV-3) using an immunoinformatics approach. To assess the immunodominant T- and B-cell epitopes, the CyHV-3 proteomes were employed. Following a thorough evaluation, we constructed a strategy for vaccination employing four possible epitopes selected from among each of the three relevant epitope groups: cytotoxic T-lymphocyte, helper T-lymphocyte and linear B-lymphocyte. Important qualities used in the evaluation of the resultant vaccine are that it will be highly soluble, antigenic, immunogenic and non-allergenic. Among acceptable physicochemical qualities, the anticipated structure of the vaccine bears a close resemblance to that of the original protein. Additional considerations include a robust and sustained predicted binding between the vaccine and the Toll-Like Receptor (TLR9). Simulations of molecular dynamics confirm the likelihood of a strong binding stability and structural tightness. Moreover, the computer-generated immunological simulation revealed that the vaccine, when administered to fish, should induce immune responses comparable to those in real life. Finally, codon optimization based on Escherichia coli K12 produced favorable indications of GC content and acceptably high CAI value, as applicable to the cloning vector pET28+ (a). Overall, these results show that the proposed peptide vaccine is a promising option for CyHV-3 prophylaxis.

Keywords: CyHV-3; Cyprinid Herpesvirus; Multi-Epitope Vaccine; Immunoinformatics; Immune Simulation; Vaccine

Introduction

Common carp (including ornamental koi carp) Cyprinus carpio L. is commercially valuable and is historically the first domesticated freshwater fish species; it is cultured extensively across the world [1-3]. This is one of the most important aquatic species raised for human consumption as well as for ornamental purposes, currently ranking 4^th among cultured fish species, representing 8.6% of the world’s finfish harvest [3]. Common carp farming has negligible environmental consequences; in 2020, 4.236 million tonnes of this fish was farmed, an increase of over 200 thousand tonnes compared to five years earlier [3,4]. This substantial increase in production, which was already at a very high level, further increases the chance of infectious diseases spreading in a farmed species [5]. The Cyprinidae family comprises a majority of farmed freshwater fish production worldwide, serving as a critically essential food source for many Asian countries. Seven of the nine fish species most abundantly cultured in 2020 are members of this family [3]. Koi herpes viruses infect common carp and ornamental koi fish, frequently resulting in mass mortality on carp farms [6]. The CyHV-3 variant of these viruses has also been found to be carried asymptomatically by other cyprinids, which could represent a greater threat to common carp and koi fish [7,8]. Moreover, the CyHV-3 strain is more dangerous due to its reactivation ability in response to environmental cues that induce stress; once infected, it persists and can inflict damage throughout the life span.

The emerging agent CyHV-3, which belongs to the family Alloherpesviridae, is the cause of fatal illnesses in koi and common carp fish. Following the dissemination of the virus, numerous symptoms have been observed in fish, including gill and internal organ necrosis, lethargy, epidermal abrasions and excessive mucus production [6]. The virus affects not only the internal organs, such as the brain, kidney tissues, liver and intestines, but also the external body. Moreover, due to a lack of immunity, secondary infections that occur after a first infection with CyHV-3 increase the risk of mortality [9].

The genome of CyHV-3 virions is contained within an icosahedral capsid, consisting of a lipid envelope and a thin, amorphous layer of protein. CyHV-3 residues are located in the transition region between the capsid and envelope. Common and koi carp are susceptible to infection with CyHV-3 transmission through gills or skin [10,11].

The common carp (Cyprinus carpio carpio) currently ranks among the most abundantly cultured fish species worldwide [3]. Some Cyprinids and a few other fishes have displayed positive PCR or in-situ hybridization results for CyHV-3, suggesting that they may be capable of transmitting viable virus as either carriers or vectors. Generally, results regarding the susceptibility of species other than Cyprinus carpio and its hybrids to CyHV-3 are indeterminate and remain subject to further clarification [12].

Vaccines are desperately needed to safeguard animal welfare and aquaculture. The Kovax company has developed live, attenuated viral vaccines for both common and koi carp, although they have only limited commercial availability [13,14]. One report cites the licensing of the Kovax vaccine as “available for use in Israel” [15]. A second live, attenuated viral vaccine against CyHV-3 has been described recently, although we have been unable to confirm its commercial availability [16]. Regardless, the aquaculture industry is very much in need of the development of effective measures for the prevention of CyHV-3.

Epitopes are critical for clinical and biological studies because they enable the development of vaccines against diverse and rapidly evolving diseases. Multi-epitopes play a significant role in the development of vaccines because they serve as precursors to diverse and rapidly evolving diseases. For in silico vaccine design, both B-cell and T-cell epitopes were utilized to evaluate potential designs for the vaccine. Initially, we chose a suitable receptor and linker for designing a vaccine that would bind to Cytotoxic T Lymphocyte (CTL), High-affinity T Lymphocyte (HTL) and Linear B Lymphocyte (LBL) epitopes [17].

Toll-Like Receptor 9 (TLR9) was used as an adjuvant in the development of a potentially successful vaccination, selected because of its efficiency at recognizing viral proteins; this quality is associated with the ability to prevent infection. TLR9 is involved in boosting the immune response and is also essential for efficient translation and synthesis [18]. Consequently, epitopes can be used to predict the immune reactions of the host, including humoral and cell-mediated immune responses. Moreover, toxicity is among the fundamental obstacles to preparing safe and effective vaccines against infection. As a result, it is necessary to optimize the vaccine before using it as an in vivo therapeutic agent [19]. The vaccine development process can be made more safe, effective and economically viable through computational design. Peptide vaccines are advantageous in comparison with traditional vaccines and can be prepared and tested for use in the next generation of vaccines. These are constructed specifically to target the immune system, minimizing or eliminating allergic effects. T-cell and B-cell epitopes have been combined to create a multi-epitope vaccine, which has been linked with an adjuvant protein to ensure it is both antigenic and allergen-free [20].

Therefore, the vaccine was docked with the immunological receptors of the vaccine in order to predictively evaluate in vivo vaccine stability. Finally, codon optimization was performed using an in silico approach to assure maximum expression in the chosen host expression system.

Material and Methods

Proteome Collection and Evaluations of Probable Proteome Antigenicity

The NCBI database was used to identify 224 potentially useful CyHV-3 proteomes for the intended vaccine development. This database is freely accessible and is popularly accepted and respected as a source of accurate protein sequence data. Viral proteome sequences were downloaded from the VaxiJen server (v2.0) for predictive assessment of antigenicity; this server uses Auto Cross-Covariance (ACC) transformation techniques and our assessments were made with a 0.525 threshold for antigenicity (VaxiJen v2.0 server [21]). Those protein sequences in this proteome with the highest antigenicity scores were selected for further study.

Predictions of CTL Epitopes and MHC Class I Alleles

Cytotoxic T lymphocytes (CTL) are immunocytes that are capable of directly destroying other infected cells. Through the viral entry process, they contribute significantly to host defensive mechanisms. The selected protein sequences were evaluated using the NetCTLv1.2 server (http://www.cbs.dtu.dk/services/NetCTL/) to predict CTL epitopes. Selected epitopes were subjected to further evaluation using the following three servers: VaxiJen v2.0, MHC class I immunogenicity (http://tools.iedb.org/immunogenicity/), ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) and AllerTop v2.0 (https://ddg-pharmfac.net/3). The default parameters were utilized for all predictions from these servers.

Prediction and Evaluation of Helper T-Lymphocyte Epitopes

Helper T-cells (HTLs) are critical in the generation of adaptive immunity because they identify foreign antigens, activating B and cytotoxic T-cells to eliminate pathogens. The MHC class II binding allele prediction tool (at http://tools.iedb.org/mhcii/) was used to identify the HTL epitopes. The antigenicity and capacity to produce interferon-c (IFNc), interleukin-4 (IL4) and interleukin-10 were used for further investigation of the anticipated epitopes (IL10). VaxiJen v2.0 was used to forecast the Antigens, while the IFNepitope, IL4pred and IL10pred servers were used with default settings to predict the IFNc, IL4 and IL10 characteristics, respectively.

Prediction and Assessment of Linear B Lymphocyte Epitopes

The induction of humoral immunity, also known as antibody-mediated immunity, relies heavily on B-cell epitopes. Antibodies and B-cells, which are a variety of leukocytes, work together to stimulate the immune system and destroy invaders. The iBCE-EL server, accessible at http://www.thegleelab.org/iBCE-EL/, was used with the default settings to predict the linear B lymphocyte epitopes. Evaluating projected LBL epitopes also involved using the VaxiJen v2.0, ToxinPred and AllerTop v2.0 servers.

Estimation of Population Coverage

To gauge a vaccine’s global efficacy, computational vaccination design analyzes the frequency of HLA (Human Leukocyte Antigen) alleles that correspond to the target epitope. HLA binding alleles for T-cell epitopes were used to determine population coverage. Selected epitopes and associated allelic data were submitted using the IEDB population coverage tool.

Peptide Modeling and Molecular Docking

We used the PEP-FOLD server v3.0 to submit our chosen CTL and HTL epitopes for modeling. A total of 200 simulations were carried out using a sOPEP sorting algorithm. The HLA-B 15:01 and HLA-C 06:02 alleles were considered for chosen CTL epitopes, whereas the DRB101:01 and DRB115:01 alleles were evaluated for chosen HTL epitopes. The HLA allele crystal structures were downloaded from the Protein Data Bank (PDB) and analyzed in BIOVIA Discovery Studio. For estimation of molecular docking capabilities, the AutoDock program constructed a grid-box around the functional location of each HLA allele. The docking properties of selected epitopes in their interactions with corresponding HLA alleles were determined using the AutoDock Vina script. The effectiveness of epitope binding was measured using the corresponding co-crystal ligands as positive controls.

The Multi-Epitope Approach to Designing a Potentially Safe, Effective Vaccine

The chosen CTL, HTL and LBL epitopes were linked to the potential vaccine design using an appropriate adjuvant and linkers. Toll-Like Receptor 9 (TLR9) was selected and utilized as an adjuvant because viral proteins recognize this receptor and it is important for the translation and synthesis of the vaccine candidate for structural adaptation. For this reason, the CCL35.2 protein was evaluated as a potential adjuvant for immunogenic improvement of the vaccine candidate. The bi-functional linker EAAAK, which divides two weakly interacting B domains with numerous helix-forming peptides, was used to connect the vaccine’s front to an adjuvant. While Gly-Pro-Gly-Pro-Gly (GPGPG) linkers were used to join the CTL of choice, Lys-Lys (KK) linkers were employed to join the LBL and Ala-Ala-Tyr (AAY) linkers were used to join the CTL. Proteasomes utilize AAY cleavage sites to improve protein stability, reduce their immunogenicity and improve epitope presentation. The bi-lysine KK linker assists with the maintenance of the vaccine construct’s independent immunogenic activity, while the glycine-proline linker of the GPGPG avoids the generation of junctional epitopes and simplifies immune processing.

Evaluation of the Physicochemical and Immune Systems

A protein’s physicochemistry provides considerable insight into its fundamental characteristics. Predictions of physicochemical features of the vaccine were carried out using the ProtParam service, allowing researchers to more fully grasp the vaccine’s core nature. We also used the VaxiJen v2.0, MHC-I immunogenicity and SOLPRO servers to assess the vaccine’s immunological qualities [22].

Secondary Structure Prediction

PSIPRED v4.0 (PSI-blast-based secondary structure prediction) and SOPMA (Self-Optimized Prediction Method with Alignment) servers were used to determine the construct’s 2D structural properties, such as alpha helix, beta turn and random coils [23]. The SOPMA server has a prediction accuracy greater than 80%. Molecular 2D structural characteristics were examined in order to ascertain the vaccine’s structural and compositional qualities.

Homology Modelling, Refining and Validating 3D Structures

The popular server I-TASSER was employed for prediction of the vaccine’s 3D structure using the constructed amino acid residues. ITASSER web produces 3D structures showing the protein’s structure and roles with a high degree of precision utilizing an advanced algorithm. A protein sequence can be predicted and determined using this web server, including the C-score, TM-score value, RMSD and top five models. The generated 3D structure was downloaded in PDB format based on the C-score. Its values range from -5 to 2, where higher values indicate a highly confident protein model. To refine the vaccine structure, the identified 3D structure was submitted to the GalaxyRefine online web-based server. CASP10 refine was used to run this webserver [24]. Vaccine structure validity was ascertained based on the Ramachandran plot score (a measure of the standard deviations from the mean value) and the Z-score value (Z-scores within the native protein range indicate acceptability of the proposed model). The Ramachandran plot was analyzed by the Rampage server which considers allowed and disallowed regions of amino acid and the Z-score plot was analyzed by the ProSA-web [25].

Molecular Docking Studies

Analysis of interactions involved in the binding of modeled proteins to receptor molecules can be accomplished through the use of molecular docking studies. Our refined vaccine model was submitted as the ligand and TLR9 protein as the immunological receptor for molecular docking evaluation using the ClusPro v2.0 server, which is accessible at https://cluspro.bu.edu/. The TLR9 protein was selected and downloaded from the PDB server. PyMOL v2.3.4 software was used to separate the protein from the attached ligand, then to remove water and other chemicals from the receptor.

Normal Mode Analysis

The dynamics and stability of the vaccine-receptor complex was simulated for critical assessment by both software- and server-based tools. The complex was submitted to the iMODS server (http://imods.chaconlab.org) for Normal Mode Analysis (NMA). This process provides estimations of eigenvalues, deformability, B-factors and elastic network models with predictive value for explanation of the likely intracellular movement of aggregate proteins.

Immune Response Simulation

The vaccine construct was uploaded to the C-IMMSIM v10.1 server (http://www.cbs.dtu.dk/services/C-ImmSim-10.1/) and responses were retrieved for careful evaluation. A minimum interval between initial and booster doses was set at 30 days, as previously described. An in silico simulation was conducted with three injections administered at intervals of 1, 84 and 168, where one time step corresponds to one hour in reality. In addition to the simulation step maximum of 300, the stimulation parameters were kept at their default values.

Codon Adaptation and In silico Simulation of Cloning

For foreign gene expression in a host organism, codon optimization must be carried out in consideration of characteristics of the host species. To adapt the codon according to the requirements, the construct was submitted to the JCat server (http://jcat.de/). In this study, we chose the widely used E. coli K12 host, in which the entire operation is carried out with avoidance of the following three criteria: (1) restriction enzyme cleavage sites, (2) prokaryotic ribosome binding sites and (3) rho-dependent termination of transcription. The adapted sequence was evaluated based on the Codon Adaptation Index (CAI) value and Guanine-Cytosine (GC) content [26]. Finally, the adapted nucleotide sequence was used for in silico cloning into the pET28a (þ) expression vector. The entire in silico cloning operation was executed in SnapGene v4.2 software.

Results

Selection of Proteins for Maximal Antigenicity

Components of the CyHV-3 proteome were used to develop a vaccine containing 224 amino acids. Proteins displaying the highest antigenicity were chosen, based on evaluation using the widely-used server Vaxijen v2.0. This process provided insights of potential utility in the prediction of appropriate therapeutic actions against the CyHV-3 virus (Table 1). These antigenicity scores were both encouraging and consistent; a Vaxijen prediction for CyHV-3 had an antigenicity score of 0.5820 and an ANTIGENpro prediction had a score of 0.5947. Sequence information for the chosen 224 amino acids (CyHV-3) protein was deposited in GenBank under the ID number AVL28465.1QJD15206.

Potential CTL Epitopes

The selected protein was predicted to contain 137 CTL epitopes, each consisting of nine amino acids (Table S1). There were 29 CTL epitopes that were antigenic, immunogenic, harmless and non-allergenic. Due to the large number of prospective epitopes, four CTL epitopes were selected for final vaccine development based on their antigenicity scores (Table 1). The C-score is the combined score that the NetCTL server provides. The four epitopes with the lowest C-scores were chosen because this is usually indicative of high surface accessibility of the corresponding region in the protein. Regions with low C-scores are more likely to interact with antibodies and immune cells, making them potentially immunogenic. Characteristics other than C-scores, such as antigenicity, conservation and stability, were also considered in the vaccine design process.

Table 1: CTL epitopes chosen for application in the final vaccine formulation.

Potential HTL Epitopes

The IEDB server was used first to identify 260 HTL epitopes, each consisting of 15 amino acids (Table S2). A total of 16 HTL epitopes were capable of triggering the evaluated cytokines, specifically IFNc, IL-4 and IL-10. On the basis of the antigenic score (Table 2), we considered incorporating the top four HTL epitopes into the final vaccine construct.

Table 2: Four HTL epitopes selected for final vaccine construction.

Potential LBL Epitopes

Our preliminary study identified 210 LBL epitopes, each 15 amino acids in length. As a result of advance assessment, fourteen epitopes stood out as having desirable antigenicity, negligible toxicity and non-allergenic characteristics (Table S3). The four most appealing LBL epitopes for vaccine design were selected based on their high antigenicity scores (Table 3).

Table 3: The LBL epitopes chosen for the final vaccine formulation.

Vaccine Construct and Basic Properties

Finally, the selected twelve epitopes were combined with linker adjuvants for formulating designated vaccines, such as AAY, GPGPG and KK linkers, respectively. The construct was rendered immunogenic beforehand by adding an adjuvant (Fig. 1). The epitopes included in the separate class are, for example, 4 CTL, 4 HDL and 4 LBL. As a result, the total number of 295 amino acids is predicted for the final vaccine candidate (Fig. 4).

Figure 1: The dark blue, red, green, purple and green rectangle boxes represent the adjuvant, CTL, HTL and LBL epitopes, respectively, that were incorporated in the vaccination designs. EAAAK linkers (green) connected the adjuvant and the first CTL epitope; AYY linkers (light purple) joined CTL epitopes; GPGPG linkers (deep purple); and KK linkers (red) joined HTL epitopes.

Evaluation of Immunological and Physicochemical Characteristics

Physicochemical properties play important roles in the construction of the vaccine included in Table 4. Our analysis revealed that the molecular weight of the vaccine was 32393.28 Da. The isoelectric point is focused on the separation and purification of vaccine, which was compiled at 9.82 in the study and other properties were documented, such as the chemical formula (C1425H2297N409O390S31), instability index (44.72), aliphatic index 61.15 and grand average of hydropathicity -0.297. Moreover, immunological potency expressed the vaccine-related effect on the host, where the antigenicity score was 0.5005. This analysis is not only of value for revealing uses, but also provides information about allergenicity and solubility (Table 4).

Table 4: Antigenic, allergenic and physical-chemical features of the prepared vaccine candidate.

Secondary structural characteristics assessed using two distinct servers include α-helix, β-strand and random coils. In the design, the SOPMA server projected 27.80% α-helix, 21.36% β-strand and 44.75% random coils. The PSIPRED server, on the other hand, predicted 431.04% α-helix, 23.47% β-strand and 45.49% random coils (Table 5).

Table 5: The secondary structural features of the vaccine construct.

Tertiary Structure, Refinement and Validation

The popular web-based server I-TASSER was used to generate five models. Firstly, models were selected based on their C-score (-2.96). The lowest C score was chosen for the preparation of the vaccine. Moreover, the model was uploaded to Ramachandra web server for confirming 84.7% regions with GDT-HA s 0.9146, RMSD value 13.2±4.1Å and Estimated TM-score = 0.38±0.13.

Figure 2: The figure shows the tertiary structure of designated vaccine. The indicated domains are color-coded in red (α-helix), yellow (β-strand) and blue (random coil).

The tertiary model was validated with RAMPAGE servers, whereas ProSA-web servers recognized errors from the vaccine. The refined vaccine model shows the 89.15% allowable regions and the 7.4% disallowable regions (Fig. 3). The crude model had a documented Z-score value of -0.61 from the vaccine and after refinement, the value was -0.54 (Fig. 3).

Figure 3: Ramachandra plot shows the allowed (green) and disallowed regions (red) (Fig A) and the z-core was documented from the refined vaccine model.

Molecular Docking Studies

The interaction between the vaccine and TLR9 was documented and is presented in Fig. 4. It was performed through the ClusPro v2.0 server. The lowest energy score was chosen from the 30 docked complexes with various poses. Model 1 was suitable for the designated vaccine by including all criteria. It was consequently selected as the most outstanding candidate vaccine-TLR9 complex, which had an energy score of -1065.1 (Table S4 and Fig. 4), a docking score of -392.94 and a confidence score of 0.992. The receptor and ligand interface residues are listed in the supplementary file (Table S5).

Figure 4: The figure displays the vaccine and receptor interaction.

Molecular Dynamics Simulation

The internal coordinates of the complex were compiled through network meta-analysis. The independent distortion was compiled through the buildup of deformability in residues and it was documented using the chain hinge method (Fig. 5). Receptor structures may be overinterpreted because they are often improved without any limits on the number of atomic B-factors that can be added (Fig. 5). The eigenvalue was estimated (1.539 e-04) from the complex, which shows that each variance was steadily decreasing (Fig. 5). For these reasons, it was concluded that the binding interaction had a compact conformation with minor fluctuations from the vaccine and TLR9 complex.

Figure 5: Molecular dynamics simulation of the vaccine-TLR9 complex. Herein, different plots show (A) deformability index (B) atomic B-factors, (C) eigenvalue and (D) NMA variance.

Immune Response Simulation

The simulated immune response to this vaccine bore similarities to established immunological responses to certain infections (Fig. 6). Initial immune responses were shown to be lower than secondary and tertiary immune responses (Fig. 6). Higher levels of antibodies, such as IgG1 + IgG2, IgM and IgG + IgM, are associated with such secondary and tertiary reactions. As a result, antigen extenuation demonstrates the activity of memory cells, leading to accelerated antigen clearance after subsequent exposures. Furthermore, the survival of B-cells, cytotoxic T-cells and helper T-cells (Fig. 6) revealed alterations in immune cells and IgM memory development. Several components (IFN, IL-4 and IL-10) are added to demonstrate higher and lower-level responses (Fig. 6). In terms of proportion (%) and number (cells/mm³), Th1 reactions were greater than Th0 reactions (6F). Finally, the study predicted dendritic cell movement as well as enhanced and protracted macrophage movement. Dendritic cells are present in tissues that are in direct touch with the external environment, including the mucosa of the lungs, the epithelial cells of the skin and the linings of the nasal cavity and the gastrointestinal tract (Fig. 6).

Figure 6: The figure illustrates properties of the proposed vaccine of Cyprinid herpesvirus-3. (A) primary, secondary and tertiary immune responses, (B) B-cell population, (C) cytotoxic T-cell population, (D) helper T-cell population, (E) induction of cytokines and interleukins, (F) Th1 mediated immune response, (G) EP Population per state (H) macrophage population per state and (I) dendritic cell population per state.

Codon Adaptation and In silico Cloning

The translation efficiency was improved for the construction of vaccines through the JCat server, which optimized the codon based on E. coli K12. The length of the designated peptide vaccine was 295 AA residues from the 885 lengths of nucleotide sequences. Furthermore, the estimated GC content was 63.50% and the calculated CAI value was 1.0 (Fig. 7). The pET28a (+) vector was used to contract designated vaccines and select restriction sites XhoI and BamHI, respectively, as start and cut points (6C). The SnapGene software was utilized for cloning vector pET28a (+), which helped in the optimization of the proposed vaccine. As a result, the ultimate designated cloning vector size as determined in our study was 5493 nucleotide base pairs (bp) (Fig. 7).

Figure 7: The average GC content (the optimal percentage is between 30 – 70%) of the improved sequence, as shown in Figure 7A. Here, Figures 7 B and C illustrate the in silico cloning of the designed vaccine into the pET-28a (+) vector.

Discussion

The results of this study led to the design of a multi-epitope vaccine against the devastating Cyprinid herpesvirus-3 (CyHV-3), based on an immunoinformatics approach. This pathogen threatens vitally important large-scale Cyprinus carpio production. Conclusions based on our analyses were tested and validated using immunoinformatics methods and the vaccine based on the thymidine kinase protein appears to have an appealing assortment of biochemical characteristics with health management potential [27]. When it comes to stopping the spread of illness, only pathogen exclusion approaches the efficacy and safety of vaccination. The protective compound to be developed needs to be effective at inducing fish immunity to illnesses that are easily spread. Here, we seek to deter a CyHV-3 pandemic by developing an epitope-based vaccination that would generate a robust immune response. The use of a strong new vaccine could also stop future pandemics. Without an efficient vaccination, however, Cyprinid herpesvirus-3 (CyHV-3) infection and transmission is lethal and notoriously difficult to manage and prevent. Furthermore, no effective immunization to manage the current crisis has been made broadly available. As a result, a novel approach to vaccine development is urgently required to help prevent economic and food-security crises.

Since thymidine kinase protein of Cyprinid herpesvirus-3 (CyHV-3) shows a key role in immune invasion as well as in fish-to-fish transmission, our purpose was to design an epitope vaccine by targeting the thymidine kinase [28]. The thymidine kinase protein was chosen and targeted as an antigenic region by the cellular and humoral immune systems. Moreover, the multiple epitopes such as CTL, HTL and LBL were selected in the design for the proposed vaccine. Four of the highest-scoring antigenic CTL, HTL and LBL epitopes were used to create the vaccine, along with the appropriate linkers. The key feature of the vaccine design was to improve the analysis of the vaccine candidate’s stability, folding and expression patterns. The EAAAK linker was used to bind the adjuvant to the CTL epitope, which results in potent cellular and humoral immune responses. It is not only used for the particular antigens but also amplifies the stability and longevity of the vaccine. Finally, it was determined that the vaccine construct contained 295 amino acid residues. In terms of recombinant vaccines, the physicochemical property of solubility is an essential consideration. A solubility evaluating tool projected the vaccine construct’s solubility in E. coli and the vaccine construct has been shown to be soluble in E. coli [29]. The theoretical PI value revealed that the constitution of the vaccine was acidic. The protein’s stability index, as proposed by server tools, indicates that it will be stable following synthesis. The vaccine was hydrophilic and thermostable, according to GRAVY and aliphatic index. The vaccine is thought to have an assortment of desirable physical properties and all of the numbers on the different quantifiable factors point to a high degree of likelihood that this vaccine could be an effective defense against CyHV-3.

Furthermore, the 3D receptor structure was validated and refined based on the C-score as well as the lowest energy score. The Z score was -0.54, which appears to be a fully acceptable model for the designated vaccine design. The peptide vaccine showed potential infection-inhibiting activity and a tight interaction between the modelled vaccine as a ligand and the TLR4 protein surface, as suggested by molecular docking between the two, with an energy score of -1065.1. In order to gain insight into the probable physical basis of the protein structure and function of biological macromolecules, molecular dynamics simulation was used. The time-dependent motion of individual atoms in a protein may be modelled using dynamic simulations. A greater degree of variation was seen in the vaccine component, but after 600 ps fluctuations abated, indicating that the modelled vaccine and bound protein are likely to be stable. The research simulated the immune system to determine the most effective immune response to the virus and the optimum behaviour and cell density characteristics for target clearance. An improved immune response, based on higher vaccination doses, activated memory B-cells and T-cells. Increased helper T-cell initiation after vaccination resulted in continued IFN- and IL-2 production. This pattern successfully mimicked the humoral immune response occurring in response to elevated levels of immunoglobulin production [29]. As a result of these efforts, the intended vaccine candidate was able to be cloned in silico into the E. coli K12 expression host using the pET28a (+) cloning vector [30]. In summary, this immunoinformatics based strategy produced design parameters for a possible new Cyprinid herpesvirus-3 (CyHV-3) multi-epitope vaccine that could be useful in the management of Cyprinus carpio health. A peptide-based vaccine such as this, if successfully developed, could provide a reasonably cost-effective means of helping to prevent the spread of this disease in carp farms. This will help protect and potentially increase the production of carp in Asia, where this is a critically important food fish and protect valuable ornamental koi specimens.

Conclusion

Potential T- and B-cell epitopes in the thymidine kinase protein of CyHV-3 were identified using a number of computational techniques and then used in an in silico design for a multi-epitope vaccine. Cyprinus carpio populations may benefit from the proposed vaccine’s immunodominant qualities. Importantly, it showed potent binding to the immunological TLR9 enzymatic protein and elicited a powerful immune response in response to CyHV-3 infection. Our results suggest that this vaccine candidate might serve as a foundation upon which to build a safe and effective vaccination program for protection against spread of the pathogenic agent of the CyHV-3 pandemic. Potential epitopes discovered here may be utilized in further research. Follow-up in vitro and in vivo studies are recommended as a next step in the testing and verification of our formulation of the vaccine as a preventative measure against CyHV-3.

Conflict of Interests

The authors have no conflict of interest to declare.

Funding

This research work was funded by institutional fund projects under grant no (IFPIP: 1349-130-1443). The authors gratefully acknowledge technical and financial support provided by Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors gratefully thank the numerous colleagues who helped to bring this new discovery to light.

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Supplementary Tables

Table S1: CTL epitopes for the vaccine construction.

Table S2: HTL epitopes for the vaccine construction.