In silico multi-epitope Bunyumwera virus vaccine to target virus nucleocapsid N protein

Background Bunyumwera virus can cause 82% mortality in humans currently with no vaccine or drugs for treatment. We described an in silico multi-epitope vaccine targeting Bunyumwera virus nucleocapsid N-protein and predicted B and T cell epitopes for immunogenicity, allergenicity, toxicity, and conservancy. For creating the most potent immunological response possible, docking epitopes with HLA alleles are chosen to screen them. The 3D vaccination was docked with the Toll-like receptor-8 using molecular dynamic simulations. To ensure production efficiency, the vaccine sequence was further cloned in silico in a plasmid pIB2 vector. For efficacy and safety, results must be supported in vitro and in vivo. Results The vaccine was cloned to enable expression and translation in a plasmid vector pIB2. It was expected to be antigenic, non-allergenic, and have a high binding affinity with TLR-8 in silico cloning. This multi-epitope vaccination may stimulate both innate and adaptive immunity. Conclusion The vaccine developed in this work was based on the nucleocapsid N-protein of the Bunyumwera virus and was created using a reverse vaccinology method. Further experimental validation is required to assess the vaccine’s therapeutic effectiveness and immunogenicity.


Background
In the Bunyaviridae family, the Bunyamwera group is one of 18 serologically discovered arbovirus serogroups in the Orthobunyavirus genus. They are made up of three single-stranded RNA segments along with nucleoproteins. Bunyamwera virus is prevalent in sub-Saharan Africa and is a leading cause of severe fever sickness in humans. The virus was identified from people in Uganda, Nigeria, and South Africa, and antibodies have been discovered in humans throughout sub-Saharan Africa, with a high frequency (up to 82%) in some places [1]. The virus was isolated from multiple Aedes species mosquitos, indicating that they are the primary carrier. Cache Valley Fever virus was recently characterized as a Bunyamwera virus strain, extending the infection's total geographic distribution to North America. Other Bunyamwera virus strains were discovered in Argentina. In humans and mammals, the Bumyamwera virus-related illness was found to induce minor symptoms such as fever, joint discomfort, and rash. The Bunyamwera virus family consists of 32 viruses; among them, the viruses have the primary host as human -Batai, Bunyamwera, Fort, Germiston, Guaroa Ilesha, Ngari, Shokwe, and Xingu [2].
Bumyaviruses have a nucleocapsid protein (NP) that aids in the encapsidation of genomic RNA and viral replication. In the form of ribonucleoprotein complexes, copies of the N protein encapsulated genomic RNA segments. The N protein is employed in many serological and molecular diagnostics because it is the most abundant in viral particles and infected cells [3]. Using silico techniques, this work aimed to create an effective epitope-based peptide vaccination based on the known nucleocapsid N protein sequence of Bunyamwera virus.

Secondary structure analysis and recovery of the target protein's amino acid sequence
The amino acid sequence of the virus's nucleocapsid protein N was obtained in FASTA format from the National Centre for Biotechnology Information (NCBI) database accession number AKX73307.

Prediction of epitope properties
The antigenicity of both B cell and T cell epitopes was predicted using VaxiJen v2.0 with a threshold of 0.4 (http:// www. ddg-pharm fac. net/ vaxij en/ VaxiJ en/ VaxiJ en. html). Both B cell and T cell epitopes were tested using AllerTOP v2.0 [9] to determine their allergenicity (https:// www. ddg-pharm fac. net/ Aller TOP/). ToxinPred8 was utilized to predict the toxicity of both B and T cell epitopes using a 10-amino-acid peptide fragment (http:// crdd. osdd. net/ ragha va/ toxin pred/). Epitope antigens that were non-allergic or toxic were utilized.

Epitope conservancy and population coverage
To assess diversity and degree of conservancy in protein sequences from multiple countries, the IEDB conservation-analysis-tool [10] was used to check epitope linear sequence conservancy of projected B and T cell epitopes. The epitopes that were found to be 100% conserved were

Vaccine sequence construction
Finally, vaccinations were developed using selected epitope sequences. BCL epitopes were developed after adjuvant, HTL epitopes, and CTL epitopes. The L7/L12 ribosomal protein was used as an adjuvant in the vaccine design to improve vaccination immunogenicity. AAY, EAAAK, KK, and GPGPG linkers were used to link the adjuvant and selected epitopes for vaccine manufacturing. The adjuvant sequence is linked by the EAAAK linker, whereas the CTL, HTL, and BCL epitopes are linked by the AAY, GPGPG, and KK linkers, respectively.

Molecular docking of vaccine with the receptor
Toll-like receptor-8 (TLR-8) is thought to have a role in the immune response to RNA viruses, according to several studies [17]. As a result, the vaccine 3D structure's was docked against TLR-8. TLR-8's X-ray diffraction structure (PDB ID: 3W3G) was acquired from Protein Data Bank with a resolution of 2.3 A0 (PDB). HawkDock

Molecular dynamic simulation of the docked complex
On an Internet server called Anisotropic network model web server 2.1, a molecular dynamics simulation of a receptor vaccination complex was performed (http:// anm. csb. pitt. edu/) [18]. Molecular dynamic simulations were used to evaluate the receptor-vaccine complex interaction's stability and investigate the physical mobility of atoms and macromolecules.

Structural analysis of target proteins
The structural nucleocapsid protein N sequence of the Bunyamwera virus was 233 amino acids long, according to NCBI. The target protein had an antigenicity score of 0.5713, a molecular weight of 26621.75 kDa, and an isoelectric point of 9.30, with 25 negatively charged and 31 positively charged residues. The average hydropathicity was −0.216, with an instability index of 28.22, and aliphatic index of 87.42, and an average instability index of 28.22. The secondary structure prediction indicated a 41.63% alpha-helix, 20.17% extended strands, 5.15% betaturn, and 33.05% random coil shape.

B cell epitope prediction
In this research, we developed 32 epitopes, eight of which were tested for peptide length >9 and 100% conservation in viruses sequenced in different countries, as shown in Table 1. Out of 8 epitopes, only two antigenic, non-allergenic, and non-toxic properties offering epitomes were selected, of which SGLGWKKTNVSA showed maximum antigenicity (1.8250).

T cell epitope prediction
With 261 peptides of 9-mer length, we identified 20 HLA class I supertypes. We chose 12 peptides with 100% conservancy and affinity for various HLA Class I alleles ( Table 2). KRSEWEVTL (1.5287) had the highest antigenicity, while HTL epitopes did not overlap with HLA epitopes. And the 4 peptides, which were antigenic, nonallergic, and non-toxic were chosen for further population coverage study.
With 89.42% global coverage (Fig. 1), 4 CTL epitopes were submitted to population coverage analysis in IEDB against their restricted MHC alleles. Europe (96.21%) had the largest population coverage, followed by North America (88.61%) and East Asia (86.88%). For the   (Table 3). In the NetMHCIIpan 3.2 server, 65 binding solid peptides were identified as possible HTL epitopes, of which 12 bound firmly to multiple HLA class II alleles with 100% conservancy (Table 4) and three were chosen for vaccine development.

Protein-peptide docking analysis
After obtaining the 3D structure of HTL and CTL epitopes from the PEPFOLD server, PyRxVina was used to undertake a molecular docking research, utilizing 5 models of each epitope created by PEPFOLD 3. Seven T cell epitopes were docked with 4U6Y and 1BX2 receptors, and ten docked poses of each epitope were examined in PyMol using HLA alleles. The epitopes' binding affinities revealed a strong interaction with their respective receptors (Table 5).

Vaccine construction, properties prediction, and structural analysis
L7/L12 ribosomal protein adjuvant is a 124 amino acid sequence used in vaccine development. The final vaccination sequence was 275 amino acids long, including 1 adjuvant, 4CTL, 3HTL, 2BCL epitopes, and numerous linkers. The proposed vaccine was projected to have an antigenicity of 0.7310, making it a possible antigen. The vaccination has been designed toward being non-allergic and non-toxic. The physicochemical parameters predicted by ProtParam were   Fig. 2A). The vaccine's tertiary structure, which was created in Phyre 2, was refined in Mod refiner saves ver. 6.0, which has 5 different parameter tools to evaluate the refined structure and the best model was chosen (Fig. 2B). Furthermore, PROCHECK's Ramachandran plot demonstrated that 96.6% of residues were in the most desired areas, 1.7% in extra allowed regions, and 0% in liberally allowed regions (Fig. 2C). ERRAT (Fig. 2D). VERIFY 3D −98.53% of the residues have averaged 3D-1D score    Fig. 7 below. Following that, molecular dynamic simulation studies on TLR-8 and vaccine docking were performed in the ANM 2.1 server. Peaks in (Fig. 8A) show B factor graphs of the receptor-ligand docked complex. Figure 9a, b shows the correlation map, whereas the covariance map shows the coupling between pairs of residues. The correlation is shown by red, non-correlation is indicated by white, and negative correlation is indicated by blue (Fig. 9). The deformation energies of both the chains were displayed in graphs (Fig. 10). Eigenvalues indicate the energy required to alter the structure: we discovered a TLR-8 and vaccine docked complex Eigenvalue 5.673205e−06 (Fig. 11).

In silico cloning
The Java Codon Adaptation Tool (JCat) optimized a codon sequence of 800 nucleotides with a codon adaptation index (CAI) of 0.99, effective vaccine expression in E. coli-K12 strain, and GC content of 45.2, resulting in favorable transcriptional translation efficiencies. The N-and C-terminal of EcoRI and BamHI restriction sites were connected using the SnapGene tool before introducing the codon sequence into the plasmid pIB2 vector, as shown in Figs. 12, 13, and 14). The plasmid was made up of 6356 base pairs after restriction cloning was used to introduce the optimal codon sequence.

Discussion
The National Institute of Allergy and Infectious Diseases classifies bunyaviruses as a category, an emerging pathogen with the potential to cause considerable morbidity and death. (https:// www. niaid. nih. gov/ resea rch/ emerg ing-infec tious-disea ses-patho gens). This virus has no vaccination or antiviral treatment. Using several CTL, HTL, and B cell epitopes in a vaccine can stimulate both humoral and cellular immune responses with fewer side effects than a single epitopebased vaccine. Using an immunoinformatic method based on the virus nucleocapsid N-protein, we produced a multi-epitope vaccine for Bunyumwera virus. Several BCL and T cell epitopes have been discovered. After screening via a variety of immunological filters, just a few antigenic epitopes were carefully selected. As an adjuvant, L7/L12 ribosomal protein was used, as well as EAAAK, AAY, GPGPG, and KK for linking. The adjuvant stimulates TLR-4 and B cell inflammatory cytokine-induced innate immunity. The vaccine was cloned to enable expression and translation in a plasmid vector PIB2. It was expected to be antigenic, non-allergenic, and have a high binding affinity with TLR-8 in silico cloning. This multi-epitope vaccination may stimulate both innate and adaptive immunity.

Conclusion
Computer modeling approaches help in the wide-scale screening of peptides with all potential HLA alleles to obtain the best peptides in a significant population. These approaches are effective in reducing the time and money spent on identifying high-specificity epitopes for vaccine design. The vaccine developed in this work was based on the nucleocapsid N-protein of the Bunyumwera virus and was created using a reverse vaccinology method. Further experimental validation is required to assess the vaccine's therapeutic effectiveness and immunogenicity.