Experimental Validation of MHC Class I and II Peptide-Based Potential Vaccine Candidates for Human Papilloma Virus Using Sprague-Dawly Models.

Human papilloma virus (HPV) causes cervical and many other cancers. Recent trend in vaccine design is shifted toward epitope-based developments that are more specific, safe, and easy to produce. In this study, we predicted eight immunogenic peptides of CD4+ and CD8+ T-lymphocytes (MHC class I and II as M1 and M2) including early proteins (E2 and E6), major (L1) and minor capsid protein (L2). Male and female Sprague Dawly rats in groups were immunized with each synthetic peptide. L1M1, L1M2, L2M1, and L2M2 induced significant immunogenic response compared to E2M1, E2M2, E6M1 and E6M2. We observed optimal titer of IgG antibodies (>1.25 g/L), interferon-γ (>64 ng/L), and granzyme-B (>40 pg/mL) compared to control at second booster dose (240 µg/500 µL). The induction of peptide-specific IgG antibodies in immunized rats indicates the T-cell dependent B-lymphocyte activation. A substantial CD4+ and CD8+ cell count was observed at 240 µg/500 µL. In male and female rats, CD8+ cell count for L1 and L2 peptide is 3000 and 3118, and CD4+ is 3369 and 3484 respectively compared to control. In conclusion, we demonstrated that L1M1, L1M2, L2M1, L2M2 are likely to contain potential epitopes for induction of immune responses supporting the feasibility of peptide-based vaccine development for HPV.

Association study of NAT2 gene polymorphism and risk of oral cancer in Southern Punjab, Pakistan.

OBJECTIVE: To investigate the single nucleotide polymorphic variations of N-acetyltransferase 2, phase-II metabolising enzyme, and associated risk factors for oral cancer. METHODS: The case-control study was conducted from November 2017 to April 2018 after approval from the Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan, and comprised oral cancer patients and healthy controls. Single nucleotide polymorphism of the N-acetyltransferase 2 gene associated with oral cancer was analysed. Factors assessed using tetra-primer amplification refractory mutation system polymerase chain reaction included age, smoking, naswar, betel leaves and nuts. RESULTS: Of the 201 subjects, 94(47%) were patients and 107(53%) were controls, while 108(54%) were aged 10-30 years. Single nucleotide polymorphism rs1208 of N-acetyltransferase 2 gene was primarily A803G and Lys268Arg, with allelic frequency of G/A. Age range 51-70 was significantly (p=0.00001) associated with the prevalence of oral cancer in terms of genotypic relationship with A803G. Substantial allelic association was found between the gene and oral cancer (p=0.006895). Smoking increased the cacner risk 7-fold (odds ratio: 7.0). CONCLUSIONS: The genetic variant of N-acetyltransferase 2 rs1208 was found to be significantly associated with oral cancer progression and development of associated risk factors.

Antitumor activity and combined inhibitory effect of ceritinib with gemcitabine in pancreatic cancer.

Pancreatic cancer (PC) is predominantly incurable and is primarily treated with gemcitabine, but drug resistance commonly develops. Thus, new medicines are needed. Ceritinib (LDK378) is a second-generation tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) with antitumor activity in various cancers. However, studies involving ceritinib for the treatment of PC are inadequate. We analyzed the combined effects of ceritinib and gemcitabine on PC and their mechanism of action. Three PC cell lines were used to evaluate the antitumor effects of ceritinib combined with gemcitabine. We analyzed cell viability using CCK-8 assays, determined apoptosis levels through flow cytometry, and analyzed autophagy and cell signaling pathways by Western blotting and tissue array analysis with samples from xenograft models. Ceritinib strongly inhibited the proliferation of PC cells in a dose-dependent manner, induced apoptosis, and inhibited autophagy and cell migration by regulating relevant factors. Ceritinib in combination with gemcitabine exhibited significant growth inhibition and additive antitumor effects in vitro. In vivo, gemcitabine and ceritinib reduced tumor size by up to 30%. In our study, ALK was shown to be highly expressed in various PC cells and tissues. Ceritinib strongly inhibited the levels of activated ALK in PC cells with subsequent effects on the downstream mediators STAT3, AKT, and ERK. In addition, ceritinib inhibited tumor progression in xenograft models. Overall, our research shows that ceritinib inhibits the ALK signaling pathway, leading to cell growth/angiogenesis inhibition in PC and the induction of apoptosis. We recommend using ceritinib as a new treatment for PC.NEW & NOTEWORTHY These data proved that ceritinib inhibits the anaplastic lymphoma kinase signaling pathway, leading to cell growth/angiogenesis inhibition and the induction of apoptosis by inhibiting STAT3, AKT, and ERK pathway in pancreatic cancer (PC). We recommend using ceritinib as a new treatment for PC.

Experimental analysis of T cell epitopes for designing liver cancer vaccine predicted by system-level immunoinformatics approach.

Liver cancer is a worldwide disease, and, currently, due to the poor prognostic and therapeutic options of liver cancer, we investigated the T cell epitopes as potential therapeutic vaccine candidates to get the benefit of experimental processes and utilize the complete ability of the immune system compared with other artificial ex vivo proliferation of T cells. Activation of T cells targets and kills several tumors, developing a strong rationale for the improvement of immunotherapeutic strategies to cancer therapy. To predict T cell epitopes for liver cancer, we designed a comprehensive immunoinformatics framework involving data mining, immunogenicity prediction, functional proteomic analysis, conservation studies, molecular modeling, and in vivo validation analysis. We found the binding affinity of antigenic peptides with major histocompatibility complex (MHC) I molecules to control the cancerous activity. Five extracellular antigenic proteins, including complement protein (C6), serotransferrin, coagulation factor XIII B, serum albumin (ALB), and prothrombin, were identified. We predicted and synthesized T cell epitopes to human leukocytes antigen-A*01:01 allele of MHC class I molecule. The hematological assay and IgG ELISA showed that C6 and ALB epitopes induced the production of lymphocytes, granulocytes, and peptide-specific IgG in immunized rats. We observed substantial high levels of granzymes B in serum samples of C6 and ALB compared with control, indicating the activity of cytotoxic T cells. We concluded that C6 and ALB are likely to contain potential epitopes for the induction of protective effector molecules, supporting the feasibility of therapeutic peptide-based vaccine for liver cancer.NEW & NOTEWORTHY We observed substantial high levels of granzymes B in serum samples of component C6 (C6) and albumin (ALB) compared with control, indicating the activity of cytotoxic T cells. We concluded that C6 and ALB are likely to contain potential epitopes for the induction of protective effector molecules, supporting the feasibility of therapeutic peptide-based vaccine for liver cancer.

Mini-Review: molecular elucidations of hutchinson-gilford progeria syndrome: A hope for managing horrors of premature aging in children.

Hutchinson-Gilford Progeria syndrome (or Progeria) is an exceptionally rare genetic disorder in children. It is caused by a rare point mutation in the lamin gene. It encodes lamin A protein, resulting in the de-shaping of nuclear membrane. This altered structure of the nuclear membrane renders the nucleus unstable. The shortened lifespan of the nucleus makes the cell liable for rapid ageing. Children are healthy by appearance when they are born but the signs appear after 12-24 months of age. Cardiovascular system is greatly affected which became a reason for the death of most of the patients of progeria. Stiffened joints disturb the bone movements; and alopecia affects the appearance of the patient. Rate of occurrence of the disease is one per four hundred thousand of people, though both sexes are equally affected.

Antibacterial activity and characterization of zinc oxide nanoparticles synthesized by microalgae.

Biosynthesis of zinc oxide nanoparticles (ZnO-NPs) using microalgae is novel and cost-effective approach. We studied production, molecular characterization, and antibacterial activity. Filtrates of isolated microalgae strain ZAA1 (MF140241), ZAA2 (MF114592) and ZAA3 (MF114594) were used. Incubation of these strains in 5mM solution of zinc nitrate was resulted in the synthesis of ZnO-NPs. Fourier-transform infrared, UV-visible spectroscopy and scanning electron microscopy were used to characterize the nanoparticles. Significant antibacterial activity of ZnO-NPs was measured against Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Citrobacter freundii. The microalgae mediated ZnO-NPs production is a successful procedure that can be used in a wide range of biomedical applications.

Protective effects of maresin 1 against inflammation in experimentally induced acute pancreatitis and related lung injury.

Severe acute pancreatitis (SAP) is an inflammatory disorder that progresses with local and systemic difficulties accompanied by a relatively high mortality rate. In recent years, maresin 1 (MaR1) has been shown to be a macrophage mediator with effective proresolving and anti-inflammatory properties that prevents the occurrence of various inflammatory conditions. The purpose of this study was to investigate the role of MaR1 in SAP and related lung injury. Experimental SAP was induced in mice with a combination of cerulean and lipopolysaccharide. MaR1 was administered 30 min before the primary injection of cerulean. Biochemical markers and histological injury scores were used to evaluate the severity of acute pancreatitis. To determine the degree of inflammation, serum cytokines and myeloperoxidase activity in pancreas and lung tissues were measured. Western blot analysis detected the activation of NF-κB. After MaR1 pretreatment, the activities of amylase, lipase, TNF-α, IL-1ß, and IL-6 were decreased in serum, and the myeloperoxidase activity both in pancreas and in lung tissues significantly decreased, whereas the activity of anti-inflammatory cytokine IL-10 in serum was increased. MaR1-pretreated mice reduced the activation of pancreatic NF-κB and decreased the severity of pancreatic and lung-related injuries. These results confirm that MaR1 alleviated inflammation of the pancreas and lung by inhibiting the activity of NF-κB in experimentally induced acute pancreatitis and exerted anti-inflammatory effects. These findings suggest that MaR1 could be a new and useful drug in the treatment of SAP.NEW & NOTEWORTHY These results provided us evidence to confirm that maresin 1 (MaR1) can alleviate inflammation of the pancreas and lung by inhibiting the activity of NF-κB in experimental induced acute pancreatitis and exerts certain anti-inflammatory effects. These findings suggest that MaR1 could be a new and useful drug in the treatment of severe acute pancreatitis.

SHORT COMMUNICATION-Anticancer activity of Ziziphus mauritiana roots against human breast cancer cell line.

This study was designed to evaluate the anticancer activity of Ziziphus mauritiana roots. The dichloromethane and methanol extracts were prepared and anticancer activity was investigated the by using MTT assay. Human breast cancer cell line (MCF-7) was used in this study. 50µg/ml of dichloromethane extract of the roots of plant exhibited significant anticancer activity (70%) against the breast cancer cell line with IC50 20.34±0.9 using doxorubicin as standard. The study indicated that Ziziphus mauritiana has anticancer activity against MCF-7 cell line.

REVIEW- Contemporary evidence on the dynamic role of probiotics in liver diseases.

Currently probiotics are considered as an emerging therapeutic strategy in the treatment of many liver disorders. The use of probiotics beyond infection of intestinal flora is a very helpful approach. The optimistic effect of probiotics has been observed in treating the hepatic cirrhosis, hepatic encephalopathy, viral hepatitis, irritable bowel syndrome, non-alcoholic fatty liver and alcoholic liver disease. The characterize mechanisms of probiotics are still unknown but may involve in, maintaining a microbial barrier against potential pathogens, reducing the production of bacterial toxins, modulating the immune system, intestinal permeability, and the inflammatory response. Its safety issues, effectiveness, food supplements as its source are still to be studied. However, studies revealed that probiotic therapy in hepatocellular carcinoma and in portal hypertension are still weak. Larger clinical studies are required before probiotics can be recommended as a treatment modality in liver diseases.

Effect of glycine: Studying memory and behavioral changes in mice.

Glycine is an important chemical mediator of nervous system that plays a vital role in memory and other neurological functions. Therefore, the effect of glycine on these traits must be studied to understand biological mechanisms of intricate neurological system. We investigated the effect of different doses of glycine on memory and behavior using 30 albino mice models (treated and control). After two weeks of glycine dosing, we performed light and dark activity and novel-object recognition (NOR) tests to assess the cognitive traits. Brain and blood samples were taken and kept at -70°C using ultra-low temperature freezer. Neurochemical estimation of blood glycine level was estimated by high-performance liquid chromatography with electrochemical detectors (HPLC-ECD). Concentration of glycine (100, 300 and 500 mg/kg) is significantly observed (p<0.01) and it changes due to physiological variations in N-methyl-Daspartate (NMDA) an important neurotransmitter for memory. We observed significant increase in serotonin metabolites including 5-hydroxy tryptophan (5-HT, p<0.05) and 5-hydroxy indole acetic acid (5-HIAA, p<0.001) levels. Similarly,effects were found in case of dopamine (DA, p<0.05) and its metabolites: 3, 4-Dihydroxyphenylacetic acid (DOPAC,p<0.001) and homovanillic acid (HVA, p<0.001). Histopathological investigation of brain tissues showed cellular clumps at cortical junctions at higher doses of glycine as compared to control. These findings revealed that dose dependent concentration of glycine can be useful for memory loss and behavior deficits.

Assessment of the risk factors of hypertension among adult & elderly group in twin cities of Pakistan.

OBJECTIVE: To estimate the prevalence of hypertension and to explore the risk factors associated with it. METHODS: In a cross-sectional study, a population based survey was conducted on inhabitants of Rawalpindi-Islamabad region, 219 individuals; aged 18 years or above were included in the study. Blood pressure was measured along with information about individual's demographic and socio-economic characteristics were obtained using a standard questionnaire.. RESULTS: Overall prevalence of hypertension was 29.22% (males: 21.9% and females: 78.1%) in individuals residing in Rawalpindi-Islamabad. High blood pressure is more associated with obesity (59.4%) and a progressive increase in hypertension was observed with increasing age. Bivariate analysis revealed that hypertension has a significant correlation (p-value<0.05) with age, gender, family status, weight and physical health. CONCLUSIONS: The study concludes that our generation is well aware about the risks and consequences of hypertension, but they still continue to make no or little effort in managing or preventing it. The factors contributing to hypertension are low physical activity, diet and lack of interest to maintain their health.

Report: Computational drug designing of newly synthesized triazoles against potential targets of methicillin resistant Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) is resistant to known antibiotics and has become a great challenge for healthcare professionals, therefore new molecules are needed to manage this situation. In this study, new lead molecules 4-Amino-5-(2-Hydroxyphenyl)-1,2,4-Triazol-3-Thione (U1) and4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol(U1A Schiff base) were synthesized by fusion method that showed promising antibacterial activity (U1A: 26mm and U1: 14mm) against MRSA.FT-IR and NMR were used for structural characterization of these derivatives and their toxicity properties were assessed by Lipinski's rule of 5. New potential drug targets of this bacterium were also identified by comparative and subtraction genomics techniques. In particular, octanoyl-[GcvH]: protein N-octanoyl transferase and phosphor mevalonate kinase were used as potential targets in AutoDock Vina studies. This study can provide a framework to find potential drug targets for other pathogenic microorganisms that can successfully be docked with compound U1 and U1A.

Report: Analgesic and anti-inflammatory activity of aqueous-methanolic extract of Aerva javanica.

The present study was aimed to investigate the analgesic and anti-inflammatory activity of aqueous methanolic extract of Aerva javanica. For measuring analgesic activity, writhing test, hot plate method and formalin test were performed and abdominal writhing was induced by intra-peritoneal injection of 0.2ml of 3% acetic acid. While in formalin test, pain was experimentally induced by injecting 25 µl of 2.5% formalin in left hind paw. In hot plate method, pain was induced thermally by keeping the animals on a hot plate with temperature of about 51°C. Anti-inflammatory activity was assessed by carrageen an induced mice paw edema. The results showed that the extract had significant analgesic activity (p<0.05- p<0.001) and anti-inflammatory activity (p<0.01-p<0.001). Therefore, it was concluded from this study that the extracts of Aerva javanica may be used against pain and inflammation.

Anti-proliferative and computational studies of two new pregnane glycosides from Desmidorchis flava.

Two new pregnane glycosides named desmiflavasides C (1) and D (2) were isolated from the sap of Desmidorchis flava (N.E.Br.) Meve & Liede and have had their structures confirmed from 1D and 2D NMR spectroscopic techniques and mass spectrometry (ESIMS). Further, the effects of desmiflavasides C (1) and D (2) on the proliferation of breast and ovarian cancer cells as well as normal breast epithelial cells in culture were examined. Interestingly, desmiflavasides C (1) and D (2) were able to cause a substantial decline in the viability of cancer cells in a concentration-dependent manner. Moreover, treatment of normal cells with compound 2 resulted in no significant growth inhibition, indicating that its cytotoxicity was selective towards cancer cells. Furthermore, the activity of compound 2 against cancer as well as normal epithelial cells was found to be similar to that of a previously reported pregnane glycoside, nizwaside (3). Molecular docking studies of desmiflavasides C (1) and D (2) and nizwaside (3) were carried out to ascertain if it was possible to predict any important binding orientations required of small molecule drug candidates with suggested protein target molecules for the purposes of being able to predict the affinity and activity to an acceptable degree by such compounds. Desmiflavaside D (2) showed a relatively good binding affinity (-22.4449kcal/mol) as compared to the other two compounds viz., nizwaside (3) (-20.0319kcal/mol), and desmiflavaside C (1) (-19.4042kcal/mol). Docking results of the three pregnane glycosides viz., 1-3 revealed that these ligand molecules can accurately interact with the target protein.

EPICOCCUM SP., AN EMERGING SOURCE OF UNIQUE BIOACTIVE METABOLITES.

Fungi are playing a vital role for producing natural products, most productive source of lead compounds in far reaching endeavor of new drug discovery. Epicoccum fungus is known for its potential to produce diverse classes of biologically active secondary metabolites. The intent of this review is to provide detailed information about biology and chemistry of Epicoccum fungus. Most of the fungus metabolites showed cytotoxic, anticancer, antimicrobial and anti-diabetic activities. The literature given encompases the details of isolation of different unusual and unique secondary metabolites, their chemical nature and biological activities find out Epicoccum spp., a potential source of lead molecules.

Prioritizing drug targets in Clostridium botulinum with a computational systems biology approach.

A computational and in silico system level framework was developed to identify and prioritize the antibacterial drug targets in Clostridium botulinum (Clb), the causative agent of flaccid paralysis in humans that can be fatal in 5 to 10% of cases. This disease is difficult to control due to the emergence of drug-resistant pathogenic strains and the only available treatment antitoxin which can target the neurotoxin at the extracellular level and cannot reverse the paralysis. This study framework is based on comprehensive systems-scale analysis of genomic sequence homology and phylogenetic relationships among Clostridium, other infectious bacteria, host and human gut flora. First, the entire 2628-annotated genes of this bacterial genome were categorized into essential, non-essential and virulence genes. The results obtained showed that 39% of essential proteins that functionally interact with virulence proteins were identified, which could be a key to new interventions that may kill the bacteria and minimize the host damage caused by the virulence factors. Second, a comprehensive comparative COGs and blast sequence analysis of these proteins and host proteins to minimize the risks of side effects was carried out. This revealed that 47% of a set of C. botulinum proteins were evolutionary related with Homo sapiens proteins to sort out the non-human homologs. Third, orthology analysis with other infectious bacteria to assess broad-spectrum effects was executed and COGs were mostly found in Clostridia, Bacilli (Firmicutes), and in alpha and beta Proteobacteria. Fourth, a comparative phylogenetic analysis was performed with human microbiota to filter out drug targets that may also affect human gut flora. This reduced the list of candidate proteins down to 131. Finally, the role of these putative drug targets in clostridial biological pathways was studied while subcellular localization of these candidate proteins in bacterial cellular system exhibited that 68% of the proteins were located in the cytoplasm, out of which 6% was virulent. Finally, this framework may serve as a general computational strategy for future drug target identification in infectious diseases.

Taguchi's experimental design for optimizing the production of novel thermostable polypeptide antibiotic from Geobacillus pallidus SAT4.

Polypeptide antimicrobials used against topical infections are reported to obtain from mesophilic bacterial species. A thermophilic Geobacillus pallidus SAT4 was isolated from hot climate of Sindh Dessert, Pakistan and found it active against Micrococcus luteus ATCC 10240, Staphylococcus aureus ATCC 6538, Bacillus subtilis NCTC 10400 and Pseudomonas aeruginosa ATCC 49189. The current experiment was designed to optimize the production of novel thermostable polypeptide by applying the Taguchi statistical approach at various conditions including the time of incubation, temperature, pH, aeration rate, nitrogen, and carbon concentrations. There were two most important factors that affect the production of antibiotic including time of incubation and nitrogen concentration and two interactions including the time of incubation/pH and time of incubation/nitrogen concentration. Activity was evaluated by well diffusion assay. The antimicrobial produced was stable and active even at 55°C. Ammonium sulphate (AS) was used for antibiotic recovery and it was desalted by dialysis techniques. The resulted protein was evaluated through SDS-PAGE. It was concluded that novel thermostable protein produced by Geobacillus pallidus SAT4 is stable at higher temperature and its production level can be improved statistically at optimum values of pH, time of incubation and nitrogen concentration the most important factors for antibiotic production.

Designing, cloning and simulation studies of cancer/testis antigens based multi-epitope vaccine candidates against cutaneous melanoma: An immunoinformatics approach.

Background: Melanoma is the most fatal kind of skin cancer. Among its various types, cutaneous melanoma is the most prevalent one. Melanoma cells are thought to be highly immunogenic due to the presence of distinct tumor-associated antigens (TAAs), which includes carcinoembryonic antigen (CEA), cancer/testis antigens (CTAs) and neo-antigens. The CTA family is a group of antigens that are only expressed in malignancies and testicular germ cells. Methods: We used integrative framework and systems-level analysis to predict potential vaccine candidates for cutaneous melanoma involving epitopes prediction, molecular modeling and molecular docking to cross-validate the binding affinity and interaction between potential vaccine agents and major histocompatibility molecules (MHCs) followed by molecular dynamics simulation, immune simulation and in silico cloning. Results: In this study, three cancer/testis antigens were targeted for immunotherapy of cutaneous melanoma. Among many CTAs that were studied for their expression in primary and malignant melanoma, NY-ESO-1, MAGE1 and SSX2 antigens are most prevalent in cutaneous melanoma. Cytotoxic and Helper epitopes were predicted, and the finest epitopes were shortlisted based on binding score. The vaccine construct was composed of the four epitope-rich domains of antigenic proteins, an appropriate adjuvant, His tag and linkers. This potential multi-epitope vaccine was further evaluated in terms of antigenicity, allergencity, toxicity and other physicochemical properties. Molecular interaction estimated through protein-protein docking unveiled good interactions characterized by favorable binding energies. Molecular dynamics simulation ensured the stability of docked complex and the predicted immune response through immune simulation revealed elevated levels of antibodies titer, cytokines, interleukins and immune cells (NK, DC and MA) population. Conclusion: The findings indicate that the potential vaccine candidates could be effective immunotherapeutic agents that modify the treatment strategies of cutaneous melanoma.

Recurrent Rhabdomyolysis Induced by a Viral Illness in a Young Patient.

Rhabdomyolysis is a syndrome caused by skeletal muscle disruption that results in the release of muscle proteins into circulation, which can lead to life-threatening systemic complications. These complications include acute kidney injury (AKI), renal failure, compartment syndrome, and disseminated intravascular coagulopathy. Patients commonly present with muscle pain, fatigue, weakness, and dark-colored urine. We present the case of a 37-year-old male who presented to the hospital with pain in the lower limbs and difficulty in mobility for the past two days after returning from Jamaica. He had a mild cold and body aches but denied any sore throat, cough, or shortness of breath (SOB). He tested negative for COVID-19. He had attended his local hospital the previous night, but due to the long waiting time, he presented to the accident and emergency department at our hospital. His physical examination was normal, and his urine was dark in color. All laboratory test results were normal, except for creatinine kinase (CK) levels >100,000 IU/L (reference: 40-320 IU/L) and an alanine transaminase (ALT) level of 376 U/L (reference: 30-130 U/L). Magnetic resonance imaging of both femurs revealed a high signal in multiple muscle compartments bilaterally on a short TI inversion recovery (STIR) sequence. Autoimmune screening results were negative. He had a similar episode last year due to COVID-19 with elevated CK levels. He received conservative treatment with IV fluids and was discharged eight days after hospital admission.

Experimental trials of predicted CD4+ and CD8+ T-cell epitopes of respiratory syncytial virus.

Background: Respiratory syncytial virus (RSV) is the most common cause of viral lower respiratory tract infections (LRTIs) in young children around the world and an important cause of LRTI in the elderly. The available treatments and FDA-approved vaccines for RSV only lessen the severity of the infection and are recommended for infants and elderly people. Methods: We focused on developing a broad-spectrum vaccine that activates the immune system to directly combat RSV. The objective of this study is to identify CD4+ and CD8+ T-cell epitopes using an immunoinformatics approach to develop RSV vaccines. The efficacy of these peptides was validated through in-vitro and in-vivo studies involving healthy and diseased animal models. Results: For each major histocompatibility complex (MHC) class-I and II, we found three epitopes of RSV proteins including F, G, and SH with an antigenic score of >0.5 and a projected SVM score of <5. Experimental validation of these peptides on female BALB/c mice was conducted before and after infection with the RSV A2 line 19f. We found that the 3RVMHCI (CD8+) epitope of the F protein showed significant results of white blood cells (19.72 × 103 cells/µl), neutrophils (6.01 × 103 cells/µl), lymphocytes (12.98 × 103 cells/µl), IgG antibodies (36.9 µg/ml), IFN-γ (86.96 ng/L), and granzyme B (691.35 pg/ml) compared to control at the second booster dose of 10 µg. Similarly, 4RVMHCII (CD4+) of the F protein substantially induced white blood cells (27.08 × 103 cells/µl), neutrophils (6.58 × 103 cells/µl), lymphocytes (16.64 × 103 cells/µl), IgG antibodies (46.13 µg/ml), IFN-γ (96.45 ng/L), and granzyme B (675.09 pg/ml). In-vitro studies showed that 4RVMHCII produced a significant level of antibodies in sera on day 45 comparable to mice infected with the virus. 4RVMHCII also induced high IFN-γ and IL-2 secretions on the fourth day of the challenge compared to the preinfectional stage. Conclusion: In conclusion, epitopes of the F protein showed considerable immune response and are suitable for further validation.