Epitope-based Vaccine Research Articles

Developing T Cell Epitope-Based Vaccines Against Infection: Challenging but Worthwhile

Developing T Cell Epitope-Based Vaccines Against Infection: Challenging but Worthwhile

In silico design of an epitope-based vaccine ensemble for fasliolopsiasis

IntroductionFasciolopsiasis, a food-borne intestinal disease is most common in Asia and the Indian subcontinent. Pigs are the reservoir host, and fasciolopsiasis is most widespread in locations where pigs are reared and aquatic plants are widely consumed. Human infection has been most commonly documented in China, Bangladesh, Southeast Asia, and parts of India. It predominates in school-age children, and significant worm burdens are not uncommon. The causal organism is Fasciolopsis buski, a giant intestinal fluke that infects humans and causes diarrhoea, fever, ascites, and intestinal blockage. The increasing prevalence of medication resistance and the necessity for an effective vaccination make controlling these diseases challenging.MethodsOver the last decade, we have achieved major advances in our understanding of intestinal fluke biology by in-depth interrogation and analysis of evolving F. buski omics datasets. The creation of large omics datasets for F. buski by our group has accelerated the discovery of key molecules involved in intestinal fluke biology, toxicity, and virulence that can be targeted for vaccine development. Finding successful vaccination antigen combinations from these huge number of genes/proteins in the available omics datasets is the key in combating these neglected tropical diseases. In the present study, we developed an in silico workflow to select antigens for composing a chimeric vaccine, which could be a significant technique for developing a fasciolopsiasis vaccine that prevents the parasite from causing serious harm.Results and discussionThis chimeric vaccine can now be tested experimentally and compared to other vaccine candidates to determine its potential influence on human health. Although the results are encouraging, additional validation is needed both in vivo and in vitro. Considering the extensive genetic data available for intestinal flukes that has expanded with technological advancements, we may need to reassess our methods and suggest a more sophisticated technique in the future for identifying vaccine molecules.

Innovative epitopes in Staphylococcal Protein-A an immuno-informatics approach to combat MDR-MRSA infections.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge in clinical environments due to its resistance to standard antibiotics. Staphylococcal Protein A (SpA), a crucial virulence factor of MRSA, undermines host immune responses, making it an attractive target for vaccine development. This study aimed to identify potential epitopes within SpA that could elicit robust immune responses, ultimately contributing to the combat against multidrug-resistant (MDR) MRSA. The SpA protein sequence was retrieved from the UniProt database, with various bioinformatics tools employed for epitope prediction. T-cell epitopes were identified using the Tepitool server, focusing on high-affinity interactions with prevalent human leukocyte antigens (HLAs). B-cell epitopes were predicted using the BepiPred tool. Predicted epitopes underwent docking studies with HLA molecules to evaluate binding properties. In-silico analyses confirmed the antigenicity, promiscuity, and non-glycosylated nature of the selected epitopes. Several T and B cell epitopes within SpA were identified, showcasing high binding affinities and extensive population coverage. A multi-epitope vaccine construct, linked by synthetic linkers and an adjuvant, was modelled, refined, and validated through various bioinformatics assessments. The vaccine candidate was subsequently docked with Toll-like receptor 4 (TLR-4) to evaluate its potential for immunogenicity. This study lays the groundwork for developing epitope-based vaccines targeting SpA in MRSA, identifying promising candidates for experimental validation and contributing to innovative immunotherapeutic strategies against MRSA infections.

In silico design of multi-epitope vaccine candidate based on structural proteins of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most common respiratory disease-causing viral agents. Swine infected with PRRSV exhibit severe respiratory symptoms and reproductive failure, leading to significant economic losses. To address this issue, inactivated and live-attenuated vaccines have been developed. However, the current commercially available PRRSV vaccines do not confer sufficient protection or have safety issues. The use of epitope-based subunit vaccines reduce safety risks by including only specific immunogenic portions of the antigens. To enhance immune protection, this study targeted multiple structural proteins of PRRSV, including GP2, GP3, GP4, GP5, membrane (M), envelope (E), GP5a, and nucleocapsid (N), to enable the discovery of novel epitopes. Thus, a reverse vaccinology approach was utilized to design a multi-epitope subunit vaccine construct against PRRSV. Using different tools, seven linear B cell, seven cytotoxic T cell, and three helper T cell epitopes were predicted to be safe, antigenic, and immunogenic. These epitopes were linked together, and a protein adjuvant, heparin-binding hemagglutinin, was added to increase the vaccine's immunogenicity. The construct was then docked to Toll-like receptor 4 (TLR4) to assess its ability to initiate the innate immune response. The final vaccine construct was determined to be antigenic, stable, non-allergenic, and soluble. Furthermore, the vaccine demonstrated stable binding to TLR4 based on coarse-grained and atomistic molecular dynamics simulations. Finally, the immune simulation of the vaccine construct showed a robust immune response against PRRSV. In this study, a candidate vaccine construct was successfully designed as a potential strategy against PRRSV.

Computational epitope-based vaccine design with bioinformatics approach; a review.

The significance of vaccine development has gained heightened importance in light of the COVID-19 pandemic. In such critical circumstances, global citizens anticipate researchers in this field to swiftly identify a vaccine candidate to combat the pandemic's root cause. It is widely recognized that the vaccine design process is traditionally both time-consuming and costly. However, a specialized subfield within bioinformatics, known as "multi-epitope vaccine design" or "reverse vaccinology," has significantly decreased the time and costs of the vaccine design process. The methodology reverses itself in this subfield and finds a potential vaccine candidate by analyzing the pathogen's genome. Leveraging the tools available in this domain, we strive to pinpoint the most suitable antigen for crafting a vaccine against our target. Once the optimal antigen is identified, the next step involves uncovering epitopes within this antigen. The immune system recognizes particular areas of an antigen as epitopes. By characterizing these crucial segments, we gain the opportunity to design a vaccine centered around these epitopes. Subsequently, after identifying and assembling the vital epitopes with the assistance of linkers and adjuvants, our vaccine candidate can be formulated. Finally, employing computational techniques, we can thoroughly evaluate the designed vaccine. This review article comprehensively covers the entire multi-epitope vaccine development process, starting from obtaining the pathogen's genome to identifying the relevant vaccine candidate and concluding with an evaluation. Furthermore, we will delve into the essential tools needed at each stage, comparing and introducing them.

Development of a broad-spectrum epitope-based vaccine against Streptococcus pneumoniae.

Streptococcus pneumoniae (SPN) is a significant pathogen causing pneumonia and meningitis, particularly in vulnerable populations like children and the elderly. Available pneumonia vaccines have limitations since they only cover particular serotypes and have high production costs. The emergence of antibiotic-resistant SPN strains further underscores the need for a new, cost-effective, broad-spectrum vaccine. Two potential vaccine candidates, CbpA and PspA, were identified, and their B-cell, CTL, and HTL epitopes were predicted and connected with suitable linkers, adjivant and PADRE sequence. The vaccine construct was found to be antigenic, non-toxic, non-allergenic, and soluble. The three-dimensional structure of the vaccine candidate was built and validated. Docking analysis of the vaccine candidate by ClusPro demonstrated robust and stable binding interactions between the MEV and toll-like receptor 4 in both humans and animals. The iMOD server and Amber v.22 tool has verified the stability of the docking complexes. GenScript server confirmed the high efficiency of cloning for the construct and in-silico cloning into the pET28a (+) vector using SnapGene, demonstrating successful translation of the epitope region. Immunological responses were shown to be enhanced by the C-IMMSIM server. This study introduced a strong peptide vaccine candidate that has the potential to contribute to the development of a rapid and cost-effective solution for combating SPN. However, experimental verification is necessary to evaluate the vaccine's effectiveness.

Immunoinformatic approach to design T cell epitope-based chimeric vaccine targeting multiple serotypes of dengue virus

The global dengue outbreak is a significant public health concern, with the World Health Organization recording over 3 million cases and a 0.04% case fatality rate until July 2023. The infection rate is anticipated to rise in vulnerable regions worldwide. While live-attenuated vaccines are the current standard, their effectiveness in certain populations is debated. Furthermore, the presence of four closely related dengue virus serotypes can lead to antibody-dependent enhancement, compromising vaccine efficacy. In response, we propose the development of a therapeutic subunit-vaccine based on epitopes from all four serotypes to induce robust cross-protective cellular immunity. Our approach involves designing a multi-epitope chimeric immunogen using the envelope protein of the dengue virus. MHC-I and MHC-II binding T-cell epitopes were selected based on their antigen processing criteria. The most potent and immunodominant epitopes for each serotype, considering immunogenicity, population coverage, and prediction scores, were combined using AAY linker peptides to create a stable multi-epitope polypeptide. Predicted to be both antigenic and non-allergenic, the protein design exhibits a stable and soluble tertiary structure with a half-life of 4.4 h in mammalian systems. In addition, we employed an agonist to toll-like receptor-4 at the N-terminal of the vaccine design to induce downstream immunostimulatory response, validated through docking and molecular dynamics simulations. This multi-epitope construct shows promise in eliciting an effective cellular immune response against all dengue virus serotypes.

Immunoinformatics investigation on pathogenic Escherichia coli proteome to develop an epitope-based peptide vaccine candidate.

Escherichia coli (E. coli), a gram-negative bacterium, quickly colonizes in the human gastrointestinal tract after birth and typically sustains a long-term, symbiotic relationship with the host. However, certain virulent strains of E. coli can cause diseases such as urinary tract infections, meningitis, and enteric disorders. The rising antibiotic resistance among these strains has heightened the urgency for an effective vaccine. This study employs immunoinformatics and a reverse vaccinology technique to identify prospective antigens and create an efficient vaccine construct. In this study, we reported the "Attaching and Effacing Protein" a novel outer-membrane protein conserved in all pathogenic E. coli strains, based on proteome screening. We developed an in silico multi-epitope vaccine that includes helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), B cell lymphocyte (BCL), and pan HLA DR-binding reactive epitope (PADRE) sequences, along with appropriate linkers and adjuvants. Machine Learning algorithms were used to evaluate antigenicity, solubility, stability, and non-allergenicity of the vaccine construct. Additionally, molecular docking analysis revealed that vaccine construct has a strong predicted binding affinity for human toll-like receptors on the cell surface. In this context, laboratory validations are necessary to demonstrate the effectiveness of the possible vaccine design that showed encouraging findings through computational validation.

T-Cell Epitope-Based Vaccines: A Promising Strategy for Prevention of Infectious Diseases.

With the development of novel vaccine strategies, T-cell epitope-based vaccines have become promising prophylactic and therapeutic tools against infectious diseases that cannot be controlled via traditional vaccines. T-cell epitope-based vaccines leverage specific immunogenic peptides to elicit protective T-cell responses against infectious pathogens. Compared to traditional vaccines, they provide superior efficacy and safety, minimizing the risk of adverse side effects. In this review, we summarized and compared the prediction and identification methods of T-cell epitopes. By integrating bioinformatic prediction and experimental validation, efficient and precise screening of T-cell epitopes can be achieved. Importantly, we delved into the development approaches to diverse T-cell epitope-based vaccines, comparing their merits and demerits, as well as discussing the prevalent challenges and perspectives in their applications. This review offers fresh perspectives for the formulation of safe and efficacious epitope-based vaccines for the devastating diseases against which no vaccines are currently available.

Design of an Epitope-Based Vaccine Against MERS-CoV.

Background and Objectives: Middle East Respiratory Syndrome (MERS) is a viral respiratory illness caused by a coronavirus called Middle East respiratory syndrome. In the current study, immunoinformatics studies were applied to design an epitope-based vaccine construct against Middle East Respiratory Syndrome. Materials and Methods: In this study, epitopes base vaccine construct was designed against MERS using immunoinformatics approach. Results: In this approach, the targeted proteins were screened, and probable antigenic, non-allergenic, and good water-soluble epitopes were selected for vaccine construction. In vaccine construction, the selected epitopes were joined by GPGPG linkers, and a linear multi-epitope vaccine was constructed. The vaccine construct underwent a physiochemical property analysis. The 3D structure of the vaccine construct was predicted and subjected to refinement. After the refinement, the 3D model was subjected to a molecular docking analysis, TLRs (TLR-3 and TLR-9) were selected as receptors for vaccine construct, and the molecular docking analysis study determined that the vaccine construct has binding ability with the targeted receptor. Conclusions: The docking analysis also unveils that the vaccine construct can properly activate immune system against the target virus however experimental validation is needed to confirm the in silico findings further.

Application of Immunoinformatics for Efficient Epitope-Based Peptide Vaccine Development

As global public health continues to face evolving challenges, developing effective and safe vaccines has become increasingly crucial, with the design of epitope-based vaccines representing a significant breakthrough in the field. The design of epitope vaccines generally involves utilizing immunoinformatics tools and techniques to analyze known or newly identified amino acid sequences, allowing for pre-selecting and identifying potential dominant epitopes. Subsequently, peptide vaccines containing these epitopes are synthesized through chemical or genetic engineering. The rapid progress in Immunoinformatics has greatly expanded its application in vaccinology, making it the most efficient approach for developing epitope-based peptide vaccines. The review outlines the general workflow of Immunoinformatics in the design and validation of epitope vaccines, highlights the key Immunoinformatics tools used in epitope vaccine development, and discusses the specific applications of Immunoinformatics in epitope vaccine design. These insights aim to provide valuable references for the rational design and development of potent epitope vaccines.

NitraTh epitope-based neoantigen vaccines for effective tumor immunotherapy

Neoantigen vaccines represent an emerging and promising strategy in the field of tumor immunotherapy. Despite their potential, designing an effective neoantigen vaccine remains a challenge due to the current limitations in predicting CD4+ T cell epitopes with high accuracy. Here, we introduce a novel approach to neoantigen vaccine design that does not rely on computational prediction of CD4+ T cell epitopes. Utilizing nitrated helper T cell epitope containing p-nitrophenylalanine, termed “NitraTh epitope,” we have successfully engineered a series of tumor neoantigen vaccines capable of eliciting robust neoantigen-specific immune responses. With the help of NitraTh epitope, even mutations with low predicted affinity for MHC class I molecules were successfully induced to elicit neoantigen-specific responses. In H22 cell allograft and patient-derived xenograft (PDX) liver cancer mouse models, the NitraTh epitope-based neoantigen vaccines significantly suppressed tumor progression. More strikingly, through single-cell sequencing we found that the NitraTh epitope-based neoantigen vaccines regulate macrophage reprogramming and modulate macrophages to decrease the levels of the immunosuppressive molecule prostaglandin E2 (PGE2), which in turn reshapes the tumor immunosuppressive microenvironment. In summary, NitraTh epitope-based neoantigen vaccines possess the dual effects of potently activating neoantigen-specific immunity and alleviating immunosuppression, potentially providing a new paradigm for the design of tumor neoantigen vaccines.

Bioinformatics Approaches in the Development of Antifungal Therapeutics and Vaccines.

Fungal infections are considered a great threat to human life and are associated with high mortality and morbidity, especially in immunocompromised individuals. Fungal pathogens employ various defense mechanisms to evade the host immune system, which causes severe infections. The available repertoire of drugs for the treatment of fungal infections includes azoles, allylamines, polyenes, echinocandins, and antimetabolites. However, the development of multidrug and pandrug resistance to available antimycotic drugs increases the need to develop better treatment approaches. In this new era of -omics, bioinformatics has expanded options for treating fungal infections. This review emphasizes how bioinformatics complements the emerging strategies, including advancements in drug delivery systems, combination therapies, drug repurposing, epitope-based vaccine design, RNA-based therapeutics, and the role of gut-microbiome interactions to combat anti-fungal resistance. In particular, we focused on computational methods that can be useful to obtain potent hits, and that too in a short period.

Ac-[K-Aib-C(3,9-Acm; 6,12-DFLC(Acm)KKESEK)]4-NH2

Chemoselective reactions have played a crucial role in the development of high-molecular-weight (>3000 Da) macromolecules with a branched architecture, particularly as peptides and epitope-based vaccines have emerged as promising tools for drug development. Based on this, in this study, we designed and synthesized the peptide macromolecule CH3CO-[K-Aib-C(3,9-CH2NHCOCH3; 6,12-DFLC(CH2NHCOCH3)KKESEK)]4-NH2 [Ac-K-Aib-C(3,9-Acm); 6,12-epitope)]4-NH2] using chemoselective thioether bond formation between the peptide carrier CPSOC (3,9 Acm) and the IAc-DFLC(Acm)KKESEK-NH2 peptide epitope. The conjugate was purified via RP-HPLC and characterized via HR-ESI-MS. The epitope D128FLCKKESEK137 derives from the lethal toxin, ammodytoxin A, from the V. ammodytes snake species, and it was synthesized using the SPPS Fmoc/tBu methodology and characterized via HR-ESI-MS and NMR techniques.

Identification of three novel linear B-cell epitopes on VP7 of African horse sickness virus using monoclonal antibodies

African horse sickness (AHS) is an acute and subacute infectious disease of equine species caused by the African horse sickness virus (AHSV). The VP7 of AHSV is a group-specific protein conserved in all serotypes and is an excellent candidate for the serological diagnosis and an AHS vaccine component. However, to date, B-cell epitopes on the AHSV VP7 recognized by humoral immune responses remain unclear. This study expressed the recombinant AHSV VP7 soluble in Escherichia coli and purified it for mouse immunization. Four monoclonal antibodies (mAbs) were screened and identified by hybridoma cell fusion, clonal purification, and immunological assays. The B-cell epitopes, recognized by monoclonal antibodies 4B5, 3G10, 3D7, and 4D6, were identified by a series of truncated overlapping peptides expressed as glutathione S-transferase (GST)-fusion proteins. The results revealed that 4B5 recognized the 124VQTGRYAGA132 motif, 3G10 recognized the 140RYYVPQGRT148 motif, while 3D7 and 4D6 recognized the 292QPINPPIFP300 motif. Amino acid sequence alignment indicated that three novel B-cell epitopes were conserved among various AHSV serotypes but unconserved in other orbiviruses, such as the bluetongue and epidemic hemorrhagic disease viruses. This study informs on the antigenic epitopes of AHSV VP7, facilitating future investigations into the serological diagnosis method and epitope-based vaccines against AHSV.

Design of Epitope-Based Vaccine Against SARS-CoV-2: An Immuno-Informatics Study

This study aimed to develop an epitope-based vaccine of SARS-CoV-2 S protein through an immuno-informatics study. The whole genome of SARS-CoV-2 sequences was obtained from the GISAID database and then trimmed to obtain the S protein sequences. The alignment was done by Clustal-W of MEGA software. Epitope prediction and modeling were performed by Discotope BepiPred and the PepFold3 web server. The allergic responses and physicochemical characteristics of predicted epitopes were analyzed using the AlgPred and ProtParam from ExPASy. Molecular docking and dynamic stimulation were performed using AutoDock Vina and YASARA. Biovia Discovery Studio 2019 was used to visualize the molecular docking results. The study predicted 3 potential epitopes, including ‘GDEVRQIAPGQTGKIADYNYKLP’ (epitope 1), ‘YTMSLGAENSVAYSNN’ (epitope 2), and ‘VNNSYECDIPI’ (epitope 3) located in the spike head specifically RBD region. The epitopes did not show an allergen reaction based on IgE epitope mapping. The suitable overexpression for the host of epitopes was mammalian cells. Only epitopes 1 and 2 were stable (instability index above 40). Epitopes 1, 2, and 3 interacted with BCR with binding affinity values -6.6, -7.8, and -7.5 kcal/mol. Epitope 2 wasere stable when interacting with the BCR. Therefore, three epitopes were predicted to have high potency as the SARS-CoV-2 epitope-based vaccine.

Design of an epitope-based peptide vaccine against Cryptococcus neoformans.

Cryptococcus neoformans is the highest-ranked fungal pathogen in the Fungal Priority Pathogens List (FPPL) released by the World Health Organization (WHO). In this study, through in silico simulations, a multi-epitope vaccine against Cryptococcus neoformans was developed using the mannoprotein antigen (MP88) as a vaccine candidate. Following the retrieval of the MP88 protein sequences, these were used to predict antigenic B-cell and T-cell epitopes via the bepipred tool and the artificial neural network, respectively. Conserved B-cell epitopes AYSTPA, AYSTPAS, PASSNCK, and DSAYPP were identified as the most promising B-cell epitopes. While YMAADQFCL, VSYEEWMNY, and FQQRYTGTF were identified as the best candidates for CD8+ T-cell epitopes; and YARLLSLNA, ISYGTAMAV, and INQTSYARL were identified as the most promising CD4+ T-cell epitopes. The vaccine construct was modeled along with adjuvant and peptide linkers and the expasy protparam tool was used to predict the physiochemical properties. According to this, the construct vaccine was predicted to be antigenic, nontoxic, nonallergenic, soluble, stable, hydrophilic, and thermostable. Furthermore, the three-dimensional structure was also used in docking analyses with Toll-like receptor (TLR4). Finally, the cDNA of vaccine was successfully cloned into the E. coli pET-28a (+) expression vector. The results presented here could contribute towards the design of an effective vaccine against Cryptococcus neoformans.

TgVax452, an epitope-based candidate vaccine targeting Toxoplasma gondii tachyzoite-specific SAG1-related sequence (SRS) proteins: immunoinformatics, structural simulations and experimental evidence-based approaches

BackgroundThe highly expressed surface antigen 1 (SAG1)-related sequence (SRS) proteins of T. gondii tachyzoites, as a widespread zoonotic parasite, are critical for host cell invasion and represent promising vaccine targets. In this study, we employed a computer-aided multi-method approach for in silico design and evaluation of TgVax452, an epitope-based candidate vaccine against T. gondii tachyzoite-specific SRS proteins.MethodsUsing immunoinformatics web-based tools, structural modeling, and static/dynamic molecular simulations, we identified and screened B- and T-cell immunodominant epitopes and predicted TgVax452’s antigenicity, stability, safety, adjuvanticity, and physico-chemical properties.ResultsThe designed protein possessed 452 residues, a MW of 44.07 kDa, an alkaline pI (6.7), good stability (33.20), solubility (0.498), and antigenicity (0.9639) with no allergenicity. Comprehensive molecular dynamic (MD) simulation analyses confirmed the stable interaction (average potential energy: 3.3799 × 106 KJ/mol) between the TLR4 agonist residues (RS09 peptide) of the TgVax452 in interaction with human TLR4, potentially activating innate immune responses. Also, a dramatic increase was observed in specific antibodies (IgM and IgG), cytokines (IFN-γ), and lymphocyte responses, based on C-ImmSim outputs. Finally, we optimized TgVax452’s codon adaptation and mRNA secondary structure for efficient expression in E. coli BL21 expression machinery.ConclusionOur findings suggest that TgVax452 is a promising candidate vaccine against T. gondii tachyzoite-specific SRS proteins and requires further experimental studies for its potential use in preclinical trials.

The Development of Epitope-Based Recombinant Protein Vaccines against SARS-CoV-2.

The COVID-19 pandemic continues to cause infections and deaths, which are attributable to the SARS-CoV-2 Omicron variant of concern (VOC). Moderna's response to the declining protective efficacies of current SARS-CoV-2 vaccines against Omicron was to develop a bivalent booster vaccine based on the Spike (S) protein from the Wuhan and Omicron BA.4/BA.5 strains. This approach, while commendable, is unfeasible in light of rapidly emerging mutated viral strains. PubMed and Google Scholar were systematically reviewed for peer-reviewed papers up to January 2024. Articles included focused on specific themes such as the clinical history of recombinant protein vaccine development against different diseases, including COVID-19, the production of recombinant protein vaccines using different host expression systems, aspects to consider in recombinant protein vaccine development, and overcoming problems associated with large-scale recombinant protein vaccine production. In silico approaches to identify conserved and immunogenic epitopes could provide broad protection against SARS-CoV-2 VOCs but require validation in animal models. The recombinant protein vaccine development platform has shown a successful history in clinical development. Recombinant protein vaccines incorporating conserved epitopes may utilize a number of expression systems, such as yeast (Saccharomyces cerevisiae), baculovirus-insect cells (Sf9 cells), and Escherichia coli (E. coli). Current multi-epitope subunit vaccines against SARS-CoV-2 utilizing synthetic peptides are unfeasible for large-scale immunizations. Recombinant protein vaccines based on conserved and immunogenic proteins produced using E. coli offer high production yields, convenient purification, and cost-effective production of large-scale vaccine quantities capable of protecting against the SARS-CoV-2 D614G strain and its VOCs.

Identification and Characterization of a Novel B Cell Epitope of ASFV Virulence Protein B125R Monoclonal Antibody.

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The 52DPLASQRDIYY62 (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV.