Proteome Exploration of Legionella pneumophila To Identify Novel Therapeutics: a Hierarchical Subtractive Genomics and Reverse Vaccinology Approach.

Legionella pneumophila is the causative agent of a severe type of pneumonia (lung infection) called Legionnaires' disease. It is emerging as an antibiotic-resistant strain day by day. Hence, identifying novel drug targets and vaccine candidates is essential to fight against this pathogen. Here, attempts were taken through a subtractive genomics approach on the complete proteome of L. pneumophila to address the challenges of multidrug resistance. A total of 2,930 proteins from L. pneumophila proteome were investigated through diverse subtractive proteomics approaches, e.g., identification of human nonhomologous and pathogen-specific essential proteins, druggability and "anti-target" analysis, subcellular localization prediction, human microbiome nonhomology screening, and protein-protein interaction studies to find out effective drug and vaccine targets. Only three fulfilled these criteria and were proposed as novel drug targets against L. pneumophila. Furthermore, outer membrane protein TolB was identified as a potential vaccine target with a better antigenicity score. Antigenicity and transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis, and a molecular docking approach were adopted to generate the most potent epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes, along with suitable adjuvant and linkers. The designed vaccine construct showed higher binding interaction with different major histocompatibility complex (MHC) molecules and human immune TLR-2 receptors with minimum deformability at the molecular level. The present study aids the development of novel therapeutics and vaccine candidates for efficient treatment and prevention of L. pneumophila infections. However, further wet-lab-based phenotypic and genomic investigations and in vivo trials are highly recommended to validate our prediction experimentally. IMPORTANCE Legionella pneumophila is a human pathogen distributed worldwide, causing Legionnaires' disease (LD), a severe form of pneumonia and respiratory tract infection. L. pneumophila is emerging as an antibiotic-resistant strain, and controlling LD is now difficult. Hence, developing novel drugs and vaccines against L. pneumophila is a major research priority. Here, the complete proteome of L. pneumophila was considered for subtractive genomics approaches to address the challenge of antimicrobial resistance. Our subtractive proteomics approach identified three potential drug targets that are promising for future application. Furthermore, a possible vaccine candidate, "outer membrane protein TolB," was proposed using reverse vaccinology analysis. The constructed vaccine candidate showed higher binding interaction with MHC molecules and human immune TLR-2 receptors at the molecular level. Overall, the present study aids in developing novel therapeutics and vaccine candidates for efficient treatment of the infections caused by L. pneumophila.

Identification of potential inhibitory analogs of metastasis tumor antigens (MTAs) using bioactive compounds: revealing therapeutic option to prevent malignancy.

The deeper understanding of metastasis phenomenon and detection of drug targets could be a potential approach to minimize cancer mortality. In this study, attempts were taken to unmask novel therapeutics to prevent metastasis and cancer progression. Initially, we explored the physiochemical, structural and functional insights of three metastasis tumor antigens (MTAs) and evaluated some plant-based bioactive compounds as potent MTA inhibitors. From 50 plant metabolites screened, isoflavone, gingerol, citronellal and asiatic acid showed maximum binding affinity with all three MTA proteins. The ADME analysis detected no undesirable toxicity that could reduce the drug likeness properties of top plant metabolites. Moreover, molecular dynamics studies revealed that the complexes were stable and showed minimum fluctuation at molecular level. We further performed ligand-based virtual screening to identify similar drug molecules using a large collection of 376,342 compounds from DrugBank. The results suggested that several structural analogs (e.g., tramadol, nabumetone, DGLA and hydrocortisone) may act as agonist to block the MTA proteins and inhibit cancer progression at early stage. The study could be useful to develop effective medications against cancer metastasis in future. Due to encouraging results, we highly recommend further in vitro and in vivo trials for the experimental validation of the findings.

Major Insights in Dynamics of Host Response to SARS-CoV-2: Impacts and Challenges.

The coronavirus disease 2019 (COVID-19), a pandemic declared by the World Health Organization on March 11, 2020, is caused by the infection of highly transmissible species of a novel coronavirus called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). As of July 25, 2021, there are 194,372,584 cases and 4,167,937 deaths with high variability in clinical manifestations, disease burden, and post-disease complications among different people around the globe. Overall, COVID-19 is manifested as mild to moderate in almost 90% of the cases and only the rest 10% of the cases need hospitalization. However, patients with older age and those having different comorbidities have made worst the pandemic scenario. The variability of pathological consequences and clinical manifestations of COVID-19 is associated with differential host-SARS-CoV-2 interactions, which are influenced by the factors that originated from the SARS-CoV-2 and the host. These factors usually include the genomic attributes and virulent factors of the SARS-CoV-2, the burden of coinfection with other viruses and bacteria, age and gender of the individuals, different comorbidities, immune suppressions/deficiency, genotypes of major histocompatibility complex, and blood group antigens and antibodies. We herein retrieved and reviewed literatures from PubMed, Scopus, and Google relevant to clinical complications and pathogenesis of COVID-19 among people of different age, sex, and geographical locations; genomic characteristics of SARS-CoV-2 including its variants, host response under different variables, and comorbidities to summarize the dynamics of the host response to SARS-CoV-2 infection; and host response toward approved vaccines and treatment strategies against COVID-19. After reviewing a large number of published articles covering different aspects of host response to SARS-CoV-2, it is clear that one aspect from one region is not working with the scenario same to others, as studies have been done separately with a very small number of cases from a particular area/region of a country. Importantly, to combat such a pandemic as COVID-19, a conclusive understanding of the disease dynamics is required. This review emphasizes on the identification of the factors influencing the dynamics of host responses to SARS-CoV-2 and offers a future perspective to explore the molecular insights of COVID-19.

Main protease inhibitors and drug surface hotspots for the treatment of COVID-19: A drug repurposing and molecular docking approach.

Here, drug repurposing and molecular docking were employed to screen approved MPP inhibitors and their derivatives to suggest a specific therapeutic agent for the treatment of COVID-19. The approved MPP inhibitors against HIV and HCV were prioritized, while RNA dependent RNA Polymerase (RdRp) inhibitor remdesivir including Favipiravir, alpha-ketoamide were studied as control groups. The target drug surface hotspot was also investigated through the molecular docking technique. Molecular dynamics was performed to determine the binding stability of docked complexes. Absorption, distribution, metabolism, and excretion analysis was conducted to understand the pharmacokinetics and drug-likeness of the screened MPP inhibitors. The results of the study revealed that Paritaprevir (-10.9 kcal/mol) and its analog (CID 131982844) (-16.3 kcal/mol) showed better binding affinity than the approved MPP inhibitors compared in this study, including remdesivir, Favipiravir, and alpha-ketoamide. A comparative study among the screened putative MPP inhibitors revealed that the amino acids T25, T26, H41, M49, L141, N142, G143, C145, H164, M165, E166, D187, R188, and Q189 are at potentially critical positions for being surface hotspots in the MPP of SARS-CoV-2. The top 5 predicted drugs (Paritaprevir, Glecaprevir, Nelfinavir, and Lopinavir) and the topmost analog showed conformational stability in the active site of the SARS-CoV-2 MP protein. The study also suggested that Paritaprevir and its analog (CID 131982844) might be effective against SARS-CoV-2. The current findings are limited to in silico analysis and lack in vivo efficacy testing; thus, we strongly recommend a quick assessment of Paritaprevir and its analog (CID 131982844) in a clinical trial.

Combination of highly antigenic nucleoproteins to inaugurate a cross-reactive next generation vaccine candidate against Arenaviridae family.

Arenaviral infections often result lethal hemorrhagic fevers, affecting primarily in African and South American regions. To date, there is no FDA-approved licensed vaccine against arenaviruses and treatments have been limited to supportive therapy. Hence, the study was employed to design a highly immunogenic cross-reactive vaccine against Arenaviridae family using reverse vaccinology approach. The whole proteome of Lassa virus (LASV), Lymphocytic Choriomeningitis virus (LCMV), Lujo virus and Guanarito virus were retrieved and assessed to determine the most antigenic viral proteins. Both T-cell and B-cell epitopes were predicted and screened based on transmembrane topology, antigenicity, allergenicity, toxicity and molecular docking analysis. The final constructs were designed using different adjuvants, top epitopes, PADRE sequence and respective linkers and were assessed for the efficacy, safety, stability and molecular cloning purposes. The proposed epitopes were highly conserved (84%-100%) and showed greater cumulative population coverage. Moreover, T cell epitope GWPYIGSRS was conserved in Junin virus (Argentine mammarenavirus) and Sabia virus (Brazilian mammarenavirus), while B cell epitope NLLYKICLSG was conserved in Machupo virus (Bolivian mammarenavirus) and Sabia virus, indicating the possibility of final vaccine construct to confer a broad range immunity in the host. Docking analysis of the refined vaccine with different MHC molecules and human immune receptors were biologically significant. The vaccine-receptor (V1-TLR3) complex showed minimal deformability at molecular level and was compatible for cloning into pET28a(+) vector of E. coli strain K12. The study could be helpful in developing vaccine to combat arenaviral infections in the future. However, further in vitro and in vivo trials using model animals are highly recommended for the experimental validation of our findings.

Identification and host response interaction study of SARS-CoV-2 encoded miRNA-like sequences: an in silico approach.

COVID-19, a global pandemic caused by an RNA virus named SARS-CoV-2 has brought the world to a standstill in terms of infectivity, casualty, and commercial plummet. RNA viruses can encode microRNAs (miRNAs) capable of modulating host gene expression, and with that notion, we aimed to predict viral miRNA like sequences of MERS-CoV, SARS-CoV and SARS-CoV-2, analyze sequence reciprocity and investigate SARS-CoV-2 encoded potential miRNA-human genes interaction using bioinformatics tools. In this study, we retrieved 206 SARS-CoV-2 genomes, executed phylogenetic analysis, and the selected reference genome (MT434792.1) exhibited about 99% similarities among the retrieved genomes. We predicted 402, 137, and 85 putative miRNAs of MERS-CoV (NC_019843.3), SARS-CoV (NC_004718.3), and SARS-CoV-2 (MT434792.1) genome, respectively. Sequence similarity was analyzed among 624 miRNAs which revealed that the predicted miRNAs of SARS-CoV-2 share a cluster with the clad of miRNAs from MERS-CoV and SARS-CoV. Only SARS-CoV-2 derived 85 miRNAs were encountered for target prediction and 29 viral miRNAs seemed to target 119 human genes. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis suggested the involvement of respective genes in various pathways and biological processes. Finally, we focused on eight putative miRNAs influencing 14 genes that are involved in the adaptive hypoxic response, neuroinvasion and hormonal regulation, and tumorigenic progression in patients with COVID-19. SARS-CoV-2 encoded miRNAs may cause misexpression of some critical regulators and facilitate viral neuroinvasion, altered hormonal axis, and tumorigenic events in the human host. However, these propositions need validation from future studies.

In silico identification of conserved miRNAs in the genome of fibre biogenesis crop Corchorus capsularis.

Corchorus capsularis, commonly known as jute occupies the leading position in the production of natural fibre alongside lower environmental threat. Small noncoding ~21 to 24 nucleotides long microRNAs play significant roles in regulating the gene expression as well as different functions in cellular growth and development. Here, the study adopted a comprehensive in silico approach to identify and characterize the conserved miRNAs in the genome of C. capsularis including functional annotation of specific gene targets. Expressed Sequence Tags (ESTs) based homology search of 3350 known miRNAs of dicotyledons were allowed against 763 non-redundant ESTs of jute genome, resulted in the prediction of 5 potential miRNA candidates belonging five different miRNA families (miR1536, miR9567-3p, miR4391, miR11300, and miR8689). The putative miRNAs were composed of 18 nucleotides having a range of -0.49 to -1.56 MFEI values and 55%-61% of (A + U) content in their pre-miRNAs. A total of 1052 gene targets of putative miRNAs were identified and their functions were extensively analyzed. Most of the gene targets were involved in plant growth, cell cycle regulation, organelle synthesis, developmental process and environmental responses. Five gene targets, namely, NAC Domain Containing Protein, WRKY DNA binding protein, 3-dehydroquinate synthase, S-adenosyl-L-Met-dependent methyl transferase and Vascular-related NAC-Domain were found to be involved in the lignin biosynthesis, phenylpropanoid pathways and secondary wall formation. The present study might accelerate the more miRNA discovery, strengthening the complete understanding of miRNAs association in the cellular basis of lignin biosynthesis towards the production of high standard jute products.

Molecular detection and analysis of virulence genes in multi-drug resistant Escherichia coli from infected broilers.

Escherichia coli associated infections are major threats in poultry industry owing to severe economic losses each year. This study was conducted to identify E. coli isolates, to evaluate their antibiotic sensitivity and to find out their virulence patterns from infected broilers of Sylhet city in Bangladesh. Using polymerase chain reaction, a total 20 isolates were identified as E. coli from 11 chickens, exhibiting symptoms like colibacillosis and/or diarrhea. All isolates were positive for type-1 fimbrial adhesion (fimH), followed by putative avian hemolysin (hlyF) in 17 isolates; while none of the isolates was amplified with intimin (eaeA). Among 10 tested antibiotics, 100% of the isolates (n = 20) showed resistance to ampicillin, amoxicillin and tetra-cycline; but they were 100% sensitive to gentamicin. Organ specific correlations of antibiotic sensitivity were obtained among the isolates through principal component analysis (PCA) and Agglomerative Hierarchical Clustering (AHC). The 16S rRNA data of two multi-drug resistant isolates revealed closed clustering with clinical E. coli strains which could be indication of their zoonotic potential. In conclusion, the results depict higher prevalence of fimH and hlyF genes and drug resistance patterns of E. coli isolates from broilers in Sylhet city of Bangladesh.

Genome based evolutionary lineage of SARS-CoV-2 towards the development of novel chimeric vaccine.

The present study aimed to predict a novel chimeric vaccine by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein, membrane protein, envelope protein and nucleocapsid protein were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. However, the in vivo and in vitro validation of suggested vaccine molecule might be ensured with wet lab trials using model animals for the implementation of the presented data.

Comprehensive genome based analysis of Vibrio parahaemolyticus for identifying novel drug and vaccine molecules: Subtractive proteomics and vaccinomics approach.

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.

Screening and druggability analysis of some plant metabolites against SARS-CoV-2: An integrative computational approach.

The sudden outbreak of novel coronavirus has caused a global concern due to its infection rate and mortality. Despite extensive research, there are still no specific drugs or vaccines to combat SARS-CoV-2 infection. Hence, this study was designed to evaluate some plant-based active compounds for drug candidacy against SARS-CoV-2 by using virtual screening methods and various computational analyses. A total of 27 plant metabolites were screened against SARS-CoV-2 main protease proteins (MPP), Nsp9 RNA binding protein, spike receptor binding domain, spike ecto-domain and HR2 domain using a molecular docking approach. Four metabolites, i.e., asiatic acid, avicularin, guajaverin, and withaferin showed maximum binding affinity with all key proteins in terms of lowest global binding energy. The crucial binding sites and drug surface hotspots were unravelled for each viral protein. The top candidates were further employed for ADME (absorption, distribution, metabolism, and excretion) analysis to investigate their drug profiles. Results suggest that none of the compounds render any undesirable consequences that could reduce their drug likeness properties. The analysis of toxicity pattern revealed no significant tumorigenic, mutagenic, irritating, or reproductive effects by the compounds. However, withaferin was comparatively toxic among the top four candidates with considerable cytotoxicity and immunotoxicity. Most of the target class by top drug candidates belonged to enzyme groups (e.g. oxidoreductases hydrolases, phosphatases). Moreover, results of drug similarity prediction revealed two approved structural analogs of Asiatic acid i.e. Hydrocortisone (DB00741) (previously used for SARS-CoV-1 and MERS) and Dinoprost-tromethamine (DB01160) from DrugBank. In addition, two other biologically active compounds, Mupirocin (DB00410) and Simvastatin (DB00641) could be an option for the treatment of viral infections. The study may pave the way to develop effective medications and preventive measure against SARS-CoV-2. Due to the encouraging results, we highly recommend further in vivo trials for the experimental validation of our findings.

Contriving a chimeric polyvalent vaccine to prevent infections caused by herpes simplex virus (type-1 and type-2): an exploratory immunoinformatic approach.

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) cause a variety of infections including oral-facial infections, genital herpes, herpes keratitis, cutaneous infection and so on. To date, FDA-approved licensed HSV vaccine is not available yet. Hence, the study was conducted to identify and characterize an effective epitope based polyvalent vaccine against both types of Herpes Simplex Virus. The selected proteins were retrieved from ViralZone and assessed to design highly antigenic epitopes by binding analyses of the peptides with MHC class-I and class-II molecules, antigenicity screening, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach. The final vaccine was constructed by the combination of top CTL, HTL and BCL epitopes from each protein along with suitable adjuvant and linkers. Physicochemical and secondary structure analysis, disulfide engineering, molecular dynamic simulation and codon adaptation were further employed to develop a unique multi-epitope peptide vaccine. Docking analysis of the refined vaccine structure with different MHC molecules and human immune TLR-2 receptor demonstrated higher interaction. Complexed structure of the modeled vaccine and TLR-2 showed minimal deformability at molecular level. Moreover, translational potency and microbial expression of the modeled vaccine was analyzed with pET28a(+) vector for E. coli strain K12 and the vaccine constructs had no similarity with entire human proteome. The study enabled design of a novel chimeric polyvalent vaccine to confer broad range immunity against both HSV serotypes. However, further wet lab based research using model animals are highly recommended to experimentally validate our findings.Communicated by Ramaswamy H. Sarma.

Immunoinformatics approaches for designing a novel multi epitope peptide vaccine against human norovirus (Norwalk virus).

Norovirus is known as a major cause of several acute gastroenteritis (AGE) outbreaks each year. A study was conducted to develop a unique multi epitope subunit vaccine against human norovirus by adopting reverse vaccinology approach. The entire viral proteome of Norwalk virus was retrieved and allowed for further in silico study to predict highly antigenic epitopes through antigenicity, transmembrane topology screening, allergenicity assessment, toxicity analysis, population coverage analysis and molecular docking approach. Capsid protein VP1 and protein VP2 were identified as most antigenic viral proteins which generated a plethora of antigenic epitopes. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, hydrophilicity, theoretical PI and structural behavior. Molecular docking analysis of the refined vaccine with different MHCs and human immune TLR8 receptor demonstrated higher binding interaction as well. Complexed structure of the modeled vaccine and TLR8 showed minimal deformability at molecular level. The designed construct was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for its heterologous cloning and expression, and sequence of vaccine constructs showed no similarity with human proteins. However, the study could initiate in vitro and in vivo studies regarding effective vaccine development against human norovirus.

Vaccinomics strategy for developing a unique multi-epitope monovalent vaccine against Marburg marburgvirus.

Marburg virus is known to cause a severe hemorrhagic fever (MHF) in both humans and non-human primates with high degree of infectivity and lethality. To date no approved treatment is available for Marburg virus infection. A study was employed to design a novel chimeric subunit vaccine against Marburg virus by adopting reverse vaccinology approach. The entire viral proteome was retrieved from UniprotKB and assessed to design highly antigenic epitopes by antigenicity screening, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach. Envelope glycoprotein (GP) and matrix protein (VP40) were identified as most antigenic viral proteins which generated a plethora of epitopes. The final vaccine was constructed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical and secondary structure of the designed vaccine was assessed to ensure its thermostability, hydrophilicity, theoretical PI and structural behaviors. Disulfide engineering, molecular dynamic simulation and codon adaptation were further employed to develop a unique multi-epitope monovalent vaccine. Docking analysis of the refined vaccine structure with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells demonstrated higher interaction. Moreover, disulfide engineering served to lessen the high mobility region of the designed vaccine in order to extend its stability. Complexed structure of the modeled vaccine and TLR8 showed minimal deformability at molecular level. Finally, translational potency and microbial expression of the modeled vaccine was analyzed with pET28a(+) vector for E. coli strain K12. However, further in vitro and in vivo investigation could be implemented for the acceptance and validation of the predicted vaccine against Marburg virus.

Reverse vaccinology approach to design a novel multi-epitope subunit vaccine against avian influenza A (H7N9) virus.

H7N9, a novel strain of avian origin influenza was the first recorded incidence where a human was transited by a N9 type influenza virus. Effective vaccination against influenza A (H7N9) is a major concern, since it has emerged as a life threatening viral pathogen. Here, an in silico reverse vaccinology strategy was adopted to design a unique chimeric subunit vaccine against avian influenza A (H7N9). Induction of humoral and cell-mediated immunity is the prime concerned characteristics for a peptide vaccine candidate, hence both T cell and B cell immunity of viral proteins were screened. Antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach were adopted to generate the most antigenic epitopes of avian influenza A (H7N9) proteome. Further, a novel subunit vaccine was designed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, h ydrophilicity, theoretical PI and structural behavior. Homology modeling, refinement and validation of the designed vaccine allowed to construct a three dimensional structure of the predicted vaccine, further employed to molecular docking analysis with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells. Moreover, disulfide engineering was employed to lessen the high mobility region of the designed vaccine in order to extend its stability. Furthermore, we investigated the molecular dynamic simulation of the modeled subunit vaccine and TLR8 complexed molecule to strengthen our prediction. Finally, the suggested vaccine was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for checking translational potency and microbial expression.