A novel mRNA multi-epitope vaccine of Acinetobacter baumannii based on multi-target protein design in immunoinformatic approach.

Acinetobacter baumannii is a gram-negative bacillus prevalent in nature, capable of thriving under various environmental conditions. As an opportunistic pathogen, it frequently causes nosocomial infections such as urinary tract infections, bacteremia, and pneumonia, contributing to increased morbidity and mortality in clinical settings. Consequently, developing novel vaccines against Acinetobacter baumannii is of utmost importance. In our study, we identified 10 highly conserved antigenic proteins from the NCBI and UniProt databases for epitope mapping. We subsequently screened and selected 8 CTL, HTL, and LBL epitopes, integrating them into three distinct vaccines constructed with adjuvants. Following comprehensive evaluations of immunological and physicochemical parameters, we conducted molecular docking and molecular dynamics simulations to assess the efficacy and stability of these vaccines. Our findings indicate that all three multi-epitope mRNA vaccines designed against Acinetobacter baumannii are promising; however, further animal studies are required to confirm their reliability and effectiveness.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping.

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

B-Cell Epitope Prediction for Antipeptide Paratopes with the HAPTIC2/HEPTAD User Toolkit (HUT).

B-cell epitope prediction is key to developing peptide-based vaccines and immunodiagnostics along with antibodies for prophylactic, therapeutic and/or diagnostic use. This entails estimating paratope binding affinity for variable-length peptidic sequences subject to constraints on both paratope accessibility and antigen conformational flexibility, as described herein for the HAPTIC2/HEPTAD User Toolkit (HUT). HUT comprises the Heuristic Affinity Prediction Tool for Immune Complexes 2 (HAPTIC2), the HAPTIC2-like Epitope Prediction Tool for Antigen with Disulfide (HEPTAD) and the HAPTIC2/HEPTAD Input Preprocessor (HIP). HIP enables tagging of residues (e.g., in hydrophobic blobs, ordered regions and glycosylation motifs) for exclusion from downstream analyses by HAPTIC2 and HEPTAD. HAPTIC2 estimates paratope binding affinity for disulfide-free disordered peptidic antigens (by analogy between flexible-ligand docking and protein folding), from terms attributed to compaction (in view of sequence length, charge and temperature-dependent polyproline-II helical propensity), collapse (disfavored by residue bulkiness) and contact (with glycine and proline regarded as polar residues that hydrogen bond with paratopes). HEPTAD analyzes antigen sequences that each contain two cysteine residues for which the impact of disulfide pairing is estimated as a correction to the free-energy penalty of compaction. All of HUT is freely accessible online ( https://freeshell.de/~badong/hut.htm ).

Identification and Validation of Peptides Specifying SARS-CoV-2 B-Cell Epitopes Eliciting Neutralizing Antibodies.

Vaccination is an effective means of inducing immune protection to prevent transmissible diseases. During the Covid-19 pandemic, immunizations using traditional and novel vaccine platforms such as the inactivated SARSCo-V-2 vaccine, adenoviral-vectored, and nucleic acid-based mRNA vaccines have been relatively successful in controlling the rates of infection and hospitalizations. Nevertheless, the danger posed by the emergence of SARS-CoV-2 variants would set the stage for the design of next generation vaccines. To overcome the lack of efficacy of current vaccines against emerging SARS-CoV-2 variants, new vaccines must be able to overcome the reduced effectiveness of the current vaccines. Since the current Covid-19 vaccines are dependent on the whole S-protein of Wuhan strain as the antigen, mutations have rendered the current Covid-19 vaccines less effective against variants of concern (VoCs). Instead of using the whole S-protein, peptide-based epitopes could be predicted using immunoinformatic approaches, simulation of the 3D structures, overlapping peptides covering the whole length of the S-protein or peptide arrays based on synthetic peptide combinatorial libraries comprising peptides recognizable by monoclonal antibodies. B-cell epitopes were predicted, and immunogenicity of peptides was validated in mice by immunizing mice with peptides conjugated to keyhole limpet hemocyanin (KLH) mixed with Montanide 51 as an adjuvant. The immunogenicity of epitopes that could elicit peptide specific IgGs was determined by peptide-based ELISA. Neutralizing activities were determined by cPass and pseudovirus-based neutralization assays.

Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.

Peptide Antibodies: Current Status.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity.

High-resolution mapping of linear epitopes from LiNTPDase2: Advancing leishmaniasis detection using optimized protein and peptide antigens.

Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.

Mapping the functional B-cell epitopes of Shigella invasion plasmid antigen D (IpaD).

Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.

Preparation and Epitope Identification of Monoclonal Antibodies against the NS6 Protein of Porcine Deltacoronavirus (PDCoV).

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that causes substantial economic losses in the swine industry worldwide. The PDCoV NS6 protein is an accessory protein that plays a pivotal role in the viral life cycle and immune evasion. However, the functions of NS6 and its role in PDCoV pathogenesis remain largely unknown. In this study, we prepared a monoclonal antibody (mAb) 5-A11 that specifically recognizes the PDCoV NS6 protein. The mAb 5-A11 exhibited high specificity for PDCoV, with no cross-reactivity with several major porcine pathogenic viruses. Furthermore, the epitope recognized by mAb 5-A11 was precisely mapped to residues 70EYGSIYGKDFI80 of the NS6 protein using Western blot analysis. Notably, this epitope is highly conserved among different PDCoV isolates. Substantial variations were observed when comparing this epitope with the corresponding regions in the NS6 proteins of other δ coronaviruses, suggesting potential differences in the structure, function, and antigenicity of their NS6 proteins. Our findings provide valuable tools and insights for further elucidating the functions of the NS6 protein and its role in PDCoV pathogenesis, as well as for developing diagnostic and therapeutic strategies against PDCoV infection.

T-Cell Epitope Mapping of SARS-CoV-2 Reveals Coordinated IFN-γ Production and Clonal Expansion of T Cells Facilitates Recovery from COVID-19.

BACKGROUND: T-cell responses can be protective or detrimental during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, the underlying mechanism is poorly understood. METHODS: In this study, we screened 144 15-mer peptides spanning the SARS-CoV-2 spike, nucleocapsid (NP), M, ORF8, ORF10, and ORF3a proteins and 39 reported SARS-CoV-1 peptides in peripheral blood mononuclear cells (PBMCs) from nine laboratory-confirmed coronavirus disease 2019 (COVID-19) patients (five moderate and four severe cases) and nine healthy donors (HDs) collected before the COVID-19 pandemic. T-cell responses were monitored by IFN-γ and IL-17A production using ELISA, and the positive samples were sequenced for the T cell receptor (TCR) ß chain. The positive T-cell responses to individual SARS-CoV-2 peptides were validated by flow cytometry. RESULTS: COVID-19 patients with moderate disease produced more IFN-γ than HDs and patients with severe disease (moderate vs. HDs, p < 0.0001; moderate vs. severe, p < 0.0001) but less IL-17A than those with severe disease (p < 0.0001). A positive correlation was observed between IFN-γ production and T-cell clonal expansion in patients with moderate COVID-19 (r = 0.3370, p = 0.0214) but not in those with severe COVID-19 (r = -0.1700, p = 0.2480). Using flow cytometry, we identified that a conserved peptide of the M protein (Peptide-120, P120) was a dominant epitope recognized by CD8+ T cells in patients with moderate disease. CONCLUSION: Coordinated IFN-γ production and clonal expansion of SARS-CoV-2-specific T cells are associated with disease resolution in COVID-19. Our findings contribute to a better understanding of T-cell-mediated immunity in COVID-19 and may inform future strategies for managing and preventing severe outcomes of SARS-CoV-2 infection.

Molecular insight into the neutralization mechanism of human-origin monoclonal antibody AH100 against Hantaan virus.

Both Old World and New World hantaviruses are transmitted through rodents and can lead to hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome in humans without the availability of specific therapeutics. The square-shaped surface spikes of hantaviruses consist of four Gn-Gc heterodimers that are pivotal for viral entry into host cells and serve as targets for the immune system. Previously, a human-derived neutralizing monoclonal antibody, AH100, demonstrated specific neutralization against the Old World hantavirus, Hantaan virus. However, the precise mode binding of this neutralizing monoclonal antibody remains unclear. In the present study, we determined the structure of the Hantaan virus Gn-AH100 antigen-binding fragment complex and identified its epitope. Crystallography revealed that AH100 targeted the epitopes on domain A and b-ribbon and E3-like domain. Epitope mapping onto a model of the higher order (Gn-Gc)4 spike revealed its localization between neighboring Gn protomers, distinguishing this epitope as a unique site compared to the previously reported monoclonal antibodies. This study provides crucial insights into the structural basis of hantavirus neutralizing antibody epitopes, thereby facilitating the development of therapeutic antibodies.IMPORTANCEHantaan virus (HTNV) poses a significant threat to humans by causing hemorrhagic fever with renal syndrome with high mortality rates. In the absence of FDA-approved drugs or vaccines, it is urgent to develop specific therapeutics. Here, we elucidated the epitope of a human-derived neutralizing antibody, AH100, by determining the HTNV glycoprotein Gn-AH100 antigen-binding fragment (Fab) complex structure. Our findings revealed that the epitopes situated on the domain A and b-ribbon and E3-like domain of the HTNV Gn head. By modeling the complex structure in the viral lattice, we propose that AH100 neutralizes the virus by impeding conformational changes of Gn protomer, which is crucial for viral entry. Additionally, sequence analysis of all reported natural isolates indicated the absence of mutations in epitope residues, suggesting the potential neutralization ability of AH100 in diverse isolates. Therefore, our results provide novel insights into the epitope and the molecular basis of AH100 neutralization.

B cell epitope mapping: The journey to better vaccines and therapeutic antibodies.

B-cell epitope mapping is an approach that can identify and characterise specific antigen binding sites of B-cell receptors and secreted antibodies. The ability to determine the antigenic clusters of amino acids bound by B-cell clones provides unprecedented detail that will aid in developing novel and effective vaccine targets and therapeutic antibodies for various diseases. Here, we discuss conventional approaches and emerging techniques that are used to map B-cell epitopes.

Identification of novel anti-CD16a antibody clones for the development of effective natural killer cell engagers.

Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.

Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

In vitro identification of neutralizing epitopes of Rhipicephalus microplus serpin 17 (RmS-17).

BACKGROUND: Rhipicephalus microplus poses a significant problem for livestock worldwide and is primarily controlled with synthetic acaricides. The continuous use of acaricides results in the selection of resistance and causes environmental harm. Vaccination presents an alternative solution to this problem, although searching for the suitable antigen is still a work in progress. Salivary proteins hold promise for inclusion in vaccine formulation due to their roles in modulating host responses, assisting blood feeding and pathogen transmission. Serpins are a class of proteinase inhibitors and are among the molecules found in tick saliva that modulate host blood coagulation, inflammation, and adaptive immune responses. Previous studies have demonstrated the potential of R. microplus serpin 17 (RmS-17) to interfere with the host's defenses, and antibodies have been shown to neutralize its effects. This makes RmS-17 an putative target for vaccine development. METHODS: Epitope mapping of RmS-17 was achieved using in silico approach combining linear B-cell epitope and antigenicity predictor. In addition, epitope mapping using overlapping peptides in an ELISA screening was used. The serpin tridimensional structure and the epitopes spatial location within the molecule were determined. Peptides were synthetized based on the predictions and used for the production of rabbit anti-sera. Purified IgG's were used to assess the antibodies capacity to neutralize RmS-17. RESULTS: Through in silico mapping, nine potential B cell epitope regions were screened, with p1RmS-17 and p2RmS-17 selected for the experiment based on antigen prediction. In the ELISA screening using overlapping peptides, eight antibody-binding regions were identified, and p3RmS-17 and p4RmS-17 were chosen. Antibodies raised against p3RmS-17 and p4RmS-17 partially neutralized RmS-17 activity. CONCLUSION: It was found that antibodies against a single epitope are sufficient to partially neutralize RmS-17 activity. These findings support the possibility of using an epitope-based vaccine for immunization against R. microplus.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Epitope Mapping of an Anti-Mouse CCR8 Monoclonal Antibody C8Mab-2 Using Flow Cytometry.

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2.

Monoclonal antibody development for early detection of ASFV I73R protein: Identification of a linear antigenic epitope.

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66. Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.

In Silico Tools for Predicting Novel Epitopes.

Identifying antigens within a pathogen is a critical task to develop effective vaccines and diagnostic methods, as well as understanding the evolution and adaptation to host immune responses. Historically, antigenicity was studied with experiments that evaluate the immune response against selected fragments of pathogens. Using this approach, the scientific community has gathered abundant information regarding which pathogenic fragments are immunogenic. The systematic collection of this data has enabled unraveling many of the fundamental rules underlying the properties defining epitopes and immunogenicity, and has resulted in the creation of a large panel of immunologically relevant predictive (in silico) tools. The development and application of such tools have proven to accelerate the identification of novel epitopes within biomedical applications reducing experimental costs. This chapter introduces some basic concepts about MHC presentation, T cell and B cell epitopes, the experimental efforts to determine those, and focuses on state-of-the-art methods for epitope prediction, highlighting their strengths and limitations, and catering instructions for their rational use.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.