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To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs. [ABSTRACT FROM AUTHOR]

Abstract Background To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). Methods In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. Results All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. Conclusion In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens. [ABSTRACT FROM AUTHOR]

Background: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). Methods: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. Results: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. Conclusion: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Allergens are substances that cause abnormal immune responses and can originate from various sources. IgE-mediated allergies are one of the most common and severe types of allergies, affecting more than 20% of the population in Western countries. Allergens can be subdivided into a limited number of families based on their structure, but this does not necessarily indicate the origin or the route of administration of the allergen, nor is the molecular basis of allergenicity clearly understood. This review examines how understanding the allergenicity of proteins involves their structural characterization and elucidates the study of conformational diversity by nuclear magnetic resonance spectroscopy. This article also discusses allergen cross-reactivity and the mechanisms by which IgE antibodies recognize and bind to allergens based on their conformational and linear epitopes. In addition, we outline how the pH, the proteolytic susceptibility and the endosomal degradation affect the outcome of allergic reactions, and how this is correlated with conformational changes and secondary structure rearrangement events. We want to emphasize the importance of considering structural diversity and dynamics, proteolytic susceptibility and pH-dependent factors to fully comprehend allergenicity.

Cow milk is a major allergenic food. The potential prevention and treatment effects of lactic acid bacteria (LAB)-fermented dairy products on allergic symptoms have garnered considerable attention. Cow milk allergy (CMA) is mainly attributed to extracellular and/or cell envelope proteolytic enzymes with hydrolysis specificity. Numerous studies have demonstrated that LAB prevents the risk of allergies by modulating the development and regulation of the host immune system. Specifically, LAB and its effectors can enhance intestinal barrier function and affect immune cells by interfering with humoral and cellular immunity. Fermentation hydrolysis of allergenic epitopes is considered the main mechanism of reducing CMA. This article reviews the linear epitopes of allergens in cow milk and the effect of LAB on these allergens and provides insight into the means of predicting allergenic epitopes by conventional laboratory analysis methods combined with molecular simulation. Although LAB can reduceCMAin several ways, the mechanism of action remains partially clarified. Therefore, this review additionally attempts to summarize the main mechanism of LAB fermentation to provide guidance for establishing an effective preventive and treatment method for CMA and serve as a reference for the screening, research, and application of LAB-based intervention. [ABSTRACT FROM AUTHOR]

Objective To evaluate the potential biological functions of non-viral neutralizing antibodies associated with linear epitopes of the SARS-CoV-2 spike protein. Methods The linear epitope S2-78 with the highest response frequency and longest antibody retention time in COVID-19 patients was selected according to previous reports. After immunizing mice with keyhole limpet hemocyain conjugated S2-78 polypeptide(KLH-S2-78), the serum anti-S2-78 IgG antibody level, mouse behavioral phenotype and the density of microglia in the brain were detected. Results KLH-S2-78 immunized mice developed high titers of anti-S2-78 IgG antibodies(the highest titer was 25 600, A450=0.305 5), a psychomotor-like behavioral phenotype with defective sensorimotor gating, impaired olfactory function, and impaired spontaneity. The density of microglia significantly increased in the cortex and hippocampus(P＜0 .05), suggesting a state of central inflammatory stress. Conclusions Anti-S2-78 IgG antibody may cause neuroinflammatory damages in the brain tissue by stimulating the host inflammatory response.

The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.

ABSTRACT: Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.

Background: Understanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants. Methods: We used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA. Results: Overall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees. Conclusions: Understanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor (EGFR) has been established as a promising therapeutic target of diverse tumors and many antibody drugs have been developed to target the third subdomain III (TSDIII) of EGFR extracellular domain. Here, by systematically examining the crystal complex structures of EGFR ETSIII domain with a variety of antibody drugs we classified the EGFR-targeted antibodies into type-I and type-II, which can recognize and interact with two distinct epitopes in EGFR ETSIII domain, namely conformational wrist epitope and linear knuckle epitope that cover discrete and continuous protein segments on the domain surface, respectively. The knuckle-to-type-II was found to have lower absolute binding energy but higher relative binding energy than wrist-to-type-I. Subsequently, an 18-mer linear peptidic mimotope K-peptide was derived from the knuckle epitope, which, however, cannot spontaneously maintain in its native ordered conformation as that in the protein context of EGFR ETSIII domain. A disulfide stapling strategy was used to cyclize and constrain the flexible (disordered) K-peptide into a desired (roughly ordered) native-like structure, which can significantly minimize the unfavorable indirect readout effect upon binding to its cognate antibody. Further binding analysis confirmed that the stapling can moderately or significantly improve the affinity of linear K-peptide to type-I antibody by ~ 2–12-folds. The designed cyclic cK-peptide[449–466] was measured to have the highest affinity, which was improved by 11.5-fold from the linear K-peptide. In addition, both the knuckle-derived linear and cyclic peptidic mimotopes exhibited a good selectivity for type-II over type-I antibodies; cyclization can improve the selectivity. [ABSTRACT FROM AUTHOR]

Background: Immune complexing of target antigen to high affinity host antibody is recognized to impact the sensitivity of commercial heartworm antigen tests. Published information describing the effect of heat on interfering canine host antibodies is lacking. Immune complex dissociation (ICD) by heat treatment of serum for samples initially testing negative for heartworm antigen increases sensitivity of commercial antigen tests, particularly for single sex or low adult infection intensities. In this study the stability and nature of the targeted epitope and mechanism of heat ICD were examined. Methods: Canine IgG was isolated using protein-A columns from serum originating from four dogs evaluated after necropsy: one dog with evidence of previously cleared infection and three dogs with confirmed heartworm infections. These dogs were expected to have an excess of antibodies based on negative antigen test and to have no or low antigen optical density, respectively, following heat treatment. Interference of antigen detection on (non-heated) positive serum was evaluated, following 1:1 mixing of antibody/PBS solutions previously heated at 25 °C, 65 °C, 75 °C, 85 °C, 95 °C and 104 °C, compared to positive serum/PBS control measured by optical density using a commercial heartworm antigen ELISA and protein quantification. Live heartworms incubated in media for 72 h provided excretory/secretory antigen for antigen stability studies following heat, endopeptidase digestion and disulfide bond reduction. Results: Mixing antigen-positive heartworm serum with antibody solutions demonstrated a significant inhibition of antigen detection for antibody solutions previously heated at 25 °C and 65 °C relative to positive serum/PBS control. Antigen detection optical density was restored at or above the control when positive serum was mixed with solutions previously heated at 75 °C, 85 °C, 95 °C and 104 °C. Significant changes occurred in protein levels for antibody solutions heated at 75 °C, 85 °C, 95 °C and 104 °C. Relative stability of antigen from live heartworms in culture was demonstrated following heat, chemical and enzymatic treatment. Conclusions: Significant changes in protein levels and antigen binding ability occurred in IgG solutions heated above 65 °C. The findings confirm heat denaturation of antibodies as the suspected mechanism of heat ICD at 104 °C for antigen diagnosis of heartworm. No significant change occurred in antigen detection following heat, chemical or enzymatic digestions supporting a heat-stable linear nature of the epitope. [ABSTRACT FROM AUTHOR]

Objective To evaluate the potential biological functions of non-viral neutralizing antibodies associated with linear epitopes of the SARS-CoV-2 spike protein. Methods The linear epitope S2-78 with the highest response frequency and longest antibody retention time in COVID-19 patients was selected according to previous reports. After immunizing mice with keyhole limpet hemocyain conjugated S2-78 polypeptide（KLH-S2-78), the serum anti-S2-78 IgG antibody level, mouse behavioral phenotype and the density of microglia in the brain were detected. Results KLH-S2-78 immunized mice developed high titers of anti-S2-78 IgG antibodies(the highest titer was 25 600, A450=0.305 5), a psychomotor-like behavioral phenotype with defective sensorimotor gating, impaired olfactory function, and impaired spontaneity. The density of microglia significantly increased in the cortex and hippocampus（P＜0.05), suggesting a state of central inflammatory stress. Conclusions Anti-S2-78 IgG antibody may cause neuroinflammatory damages in the brain tissue by stimulating the host inflammatory response. [ABSTRACT FROM AUTHOR]

BackgroundUnderstanding the humoral immune response towards viral infection and vaccination is instrumental in developing therapeutic tools to fight and restrict the viral spread of global pandemics. Of particular interest are the specificity and breadth of antibody reactivity in order to pinpoint immune dominant epitopes that remain immutable in viral variants.MethodsWe used profiling with peptides derived from the Spike surface glycoprotein of SARS-CoV-2 to compare the antibody reactivity landscapes between patients and different vaccine cohorts. Initial screening was done with peptide microarrays while detailed results and validation data were obtained using peptide ELISA.ResultsOverall, antibody patterns turned out to be individually distinct. However, plasma samples of patients conspicuously recognized epitopes covering the fusion peptide region and the connector domain of Spike S2. Both regions are evolutionarily conserved and are targets of antibodies that were shown to inhibit viral infection. Among vaccinees, we discovered an invariant Spike region (amino acids 657-671) N-terminal to the furin cleavage site that elicited a significantly stronger antibody response in AZD1222- and BNT162b2- compared to NVX-CoV2373-vaccinees.ConclusionsUnderstanding the exact function of antibodies recognizing amino acid region 657-671 of SARS-CoV-2 Spike glycoprotein and why nucleic acid-based vaccines elicit different responses from protein-based ones will be helpful for future vaccine design.

Abstract Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), distributed widely in the poultry farms in China. Hexon is one of the major capsid proteins associated with viral species or serotypes. However, the epitopes of Hexon protein remain largely unknown. In this study, a monoclonal antibody (mAb) specific to Hexon protein of FAdV-4, designated as 3G8, was generated. Subsequently, the linear peptide recognized by 3G8 was mapped and identified as 213AYGAYVK219 using a series of overlapping peptides generated from Hexon protein. Amino acid sequence analysis revealed that the epitope recognized by 3G8 was highly conserved across all the FAdVs. The epitope was immunogenic and could be recognized by FAdV-4 positive chicken serum samples. These findings will enrich our knowledge regarding the epitope on Hexon and provide valuable information for further characterization of the antigenicity of Hexon protein.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

African swine fever (ASF) is an acute and devastating infectious disease that has caused significant economic losses to the global pig industry since it was first discovered and reported. African swine fever virus (ASFV) has a large genome encoding more than 160 proteins. The biological characteristics and functions of its various proteins still remain unclear; therefore, the efficacy of specific drugs and vaccines against ASFV remains limited. ASFV pCP312R is an important ASFV protein that exhibits good immunogenicity. In this study, five monoclonal antibodies (mAbs) targeting pCP312R were successfully prepared. Confocal microscopy observations showed that pCP312R was located in the viral factory at the late stage of ASFV infection, and was co-located with p30 and pK205R. These results suggested that pCP312R might be involved in ASFV assembly. Neutralization tests revealed that pCP312R mAb could not neutralize ASFV. Next, we identified the B cell epitopes of one of the most immunogenic mAbs and found a novel epitope of pCP312R, 72 TIPPSTDEEVIR 83 , which was conserved in different pCP312R strains. Overall, five ASFV pCP312R monoclonal antibodies were prepared, and the antigenic epitope of one strain was identified in this study, laying a foundation for further studies on ASFV pCP312R function and facilitating serological diagnosis vaccine development for ASFV., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antibody recognition of antigens is a critical element of adaptive immunity. One key class of antibody-antigen complexes is comprised of antibodies targeting linear epitopes of proteins, which in some cases are conserved elements of viruses and pathogens of relevance for vaccine design and immunotherapy. Here we report a detailed analysis of the structural and interface features of this class of complexes, based on a set of nearly 200 nonredundant high resolution antibody-peptide complex structures that were assembled from the Protein Data Bank. We found that antibody-bound peptides adopt a broad range of conformations, often displaying limited secondary structure, and that the same peptide sequence bound by different antibodies can in many cases exhibit varying conformations. Propensities of contacts with antibody loops and extent of antibody binding conformational changes were found to be broadly similar to those for antibodies in complex with larger protein antigens. However, antibody-peptide interfaces showed lower buried surface areas and fewer hydrogen bonds than antibody-protein antigen complexes, while calculated binding energy per buried interface area was found to be higher on average for antibody-peptide interfaces, likely due in part to a greater proportion of buried hydrophobic residues and higher shape complementarity. This dataset and these observations can be of use for future studies focused on this class of interactions, including predictive computational modeling efforts and the design of antibodies or epitope-based vaccine immunogens. [ABSTRACT FROM AUTHOR]

Chicken serum albumin, i.e., hen egg alpha-livetin, is a recognized food allergen in chicken meat and hen eggs. Currently, there is no immunoassay available for its detection from food matrices. The characterization of chicken serum albumin-specific antibodies and the extraction of the target protein are essential for immunoassay development. One monoclonal antibody (mAb), 3H4, was used in this study due to its selectivity to a linear epitope on avian serum albumin. To study the extraction of chicken serum albumin, phosphate-buffered saline (PBS) with two additives, i.e., sodium dodecyl sulfate (SDS) and dithiothreitol (DTT), was used for its extraction from chicken blood plasma and hen egg yolk. SDS and DTT improved the chicken serum albumin's recovery and enhanced chicken serum albumin's immunodetection. In addition, chicken serum albumin retained the best solubility and immunoreactivity after heat treatment in a neutral condition. It experienced degradation and aggregation in acidic and alkaline conditions, respectively. Overall, PBS containing 0.1% SDS and 1 mM DTT (pH 7.2) was a better extraction buffer for chicken serum albumin. However, the complexity of the food matrix and elevated temperature could reduce its solubility and immunoreactivity. [ABSTRACT FROM AUTHOR]

Antibody recognition of antigens is a critical element of adaptive immunity. One key class of antibody-antigen complexes is comprised of antibodies targeting linear epitopes of proteins, which in some cases are conserved elements of viruses and pathogens of relevance for vaccine design and immunotherapy. Here we report a detailed analysis of the structural and interface features of this class of complexes, based on a set of nearly 200 nonredundant high resolution antibody-peptide complex structures that were assembled from the Protein Data Bank. We found that antibody-bound peptides adopt a broad range of conformations, often displaying limited secondary structure, and that the same peptide sequence bound by different antibodies can in many cases exhibit varying conformations. Propensities of contacts with antibody loops and extent of antibody binding conformational changes were found to be broadly similar to those for antibodies in complex with larger protein antigens. However, antibody-peptide interfaces showed lower buried surface areas and fewer hydrogen bonds than antibody-protein antigen complexes, while calculated binding energy per buried interface area was found to be higher on average for antibody-peptide interfaces, likely due in part to a greater proportion of buried hydrophobic residues and higher shape complementarity. This dataset and these observations can be of use for future studies focused on this class of interactions, including predictive computational modeling efforts and the design of antibodies or epitope-based vaccine immunogens.

Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Allergic diseases affect many individuals world-wide and are dependent on the interaction between allergens and antibodies of the IgE isotype. Allergen-specific immunotherapy (AIT) can alter the development of the disease, e.g., through induction of allergen-specific IgG that block allergen-IgE interactions. The knowledge of epitopes recognized by allergy-causing and protective antibodies are limited. Therefore, we developed an allergome-wide peptide microarray, aiming to track linear epitope binding patterns in allergic diseases and during AIT. Here, we focused on immune responses to grass pollen allergens and found that such epitopes were commonly recognized before initiation of AIT and that AIT commonly resulted in increased antibody production against additional epitopes already after 1 year of treatment. The linear epitope binding patterns were highly individual, both for subjects subjected to and for individuals not subjected to AIT. Still, antibodies against some linear epitopes were commonly developed during AIT. For example, the two rigid domains found in grass pollen group 5 allergens have previously been associated to a diversity of discontinuous epitopes. Here, we present evidence that also the flexible linker, connecting these domains, contains regions of linear epitopes against which antibodies are developed during AIT. We also describe some commonly recognized linear epitopes on Phl p 2 and suggest how antibodies against these epitopes may contribute to or prevent allergy in relation to a well-defined stereotyped/public IgE response against the same allergen. Finally, we identify epitopes that induce cross-reactive antibodies, but also antibodies that exclusively bind one of two highly similar variants of a linear epitope. Our findings highlight the complexity of antibody recognition of linear epitopes, with respect to both the studied individuals and the examined allergens. We expect that many of the findings in this study can be generalized also to discontinuous epitopes and that allergen peptide microarrays provide an important tool for enhancing the understanding of allergen-specific antibodies in allergic disease and during AIT.

Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), distributed widely in the poultry farms in China. Hexon is one of the major capsid proteins associated with viral species or serotypes. However, the epitopes of Hexon protein remain largely unknown. In this study, a monoclonal antibody (mAb) specific to Hexon protein of FAdV-4, designated as 3G8, was generated. Subsequently, the linear peptide recognized by 3G8 was mapped and identified as 213AYGAYVK219 using a series of overlapping peptides generated from Hexon protein. Amino acid sequence analysis revealed that the epitope recognized by 3G8 was highly conserved across all the FAdVs. The epitope was immunogenic and could be recognized by FAdV-4 positive chicken serum samples. These findings will enrich our knowledge regarding the epitope on Hexon and provide valuable information for further characterization of the antigenicity of Hexon protein. [ABSTRACT FROM AUTHOR]

Cow milk protein is one of the leading food allergens. This study aimed to develop an effective method for reducing milk sensitization by evaluating antigenicity of fermented skim milk protein using Lactobacillus helveticus KLDS 1.8701, Lactobacillus plantarum KLDS 1.0386, and a combination of both strains. The proteolytic systems of strains in terms of genotype and phenotype are characterized by complete genome sequence, and evaluation the antigenicity of skim milk proteins was determined by ELISA and liquid chromatography with tandem mass spectrometry. Our results showed that the genomes encoded a variety of peptidase genes. For fermented skim milk, the degree of hydrolysis of the combined strains was higher than that of individual strain. Electrophoresis showed that the band color density of α-casein (α-CN) by fermentation of the combined strains was reduced when compared with control group. The fermentation process of the combined strains inhibited α-CN, β-lactoglobulin, and α-lactalbumin antigenicity by 69.13, 36.10, and 20.92, respectively. Major allergic epitopes of α-CN and β-lactoglobulin were cleaved by abundant proteases of combined strains. In all, this study showed that the fermentation process involving both L. helveticus and L. plantarum strains could reduce cow milk protein allergenicity through the combination of cell-envelope proteinase and peptidase on α-CN. [ABSTRACT FROM AUTHOR]

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

Effective reduction of the allergenicity of instant soy milk powder (ISMP) is practically valuable for expanding its applications. This study optimized the enzymolysis technology of ISMP using single-factor experiments and response surface methodology, combined serological analysis, cellular immunological models, bioinformatics tools, and multiple spectroscopy techniques to investigate the effects of alcalase hydrolysis on allergenicity, spatial conformation, and linear epitopes of ISMP. Under the optimal process, special IgE and IgG1 binding abilities and allergenic activity to induce cell degranulation of alcalase-hydrolyzed ISMP were reduced by (64.72 ± 1.76)%, (56.79 ± 3.72)%, and (73.3 ± 1.19)%, respectively ( P < 0.05). Moreover, the spatial conformation of instant soy milk powder hydrolysates (ISMPH) changed, including decreased surface hydrophobicity, a weaker peak of amide II band, lower contents of α-helix and β-sheet, and an enhanced content of random coil. Furthermore, the linear epitopes of major soy allergens, 9 from glycinin and 13 from β-conglycinin, could be directionally disrupted by alcalase hydrolysis. Overall, the structure-activity mechanism of alcalase hydrolysis to reduce ISMP allergenicity in vitro was preliminarily clarified. It provided a new research direction for the breakthrough in the desensitization of ISMP and a theoretical basis for revealing the potential mechanism of alcalase enzymolysis to reduce the allergenicity of ISMP.

Abstract Background The Iridoviridae family is categorized into five genera and clustered into two subfamilies: Alphairidovirinae includes Lymphocystivirus, Ranavirus (GIV), and Megalocystivirus (TGIV), which infect vertebrate hosts and Betairidovirinae includes Iridovirus and Chloriridovirus, which infect invertebrate hosts. Clustered Iridoviridae subfamilies possess host-specific characteristics, which can be considered as exclusive features for in-silico prediction of effective epitopes for vaccine development. A voting mechanism-based linear epitope (LE) prediction system was applied to identify and endorse LE candidates with a minimum length requirement for each clustered subfamily Results The experimental results showed that four conserved epitopes among the Iridovirideae family, one exclusive epitope for invertebrate subfamily and two exclusive epitopes for vertebrate family were predicted. These predicted LE candidates were further validated by ELISA assays for evaluating the strength of antigenicity and cross antigenicity. The conserved LEs for Iridoviridae family reflected high antigenicity responses for the two subfamilies, while exclusive LEs reflected high antigenicity responses only for the host-specific subfamily Conclusions Host-specific characteristics are important features and constraints for effective epitope prediction. Our proposed voting mechanism based system provides a novel approach for in silico LE prediction prior to vaccine development, and it is especially powerful for analyzing antigen sequences with exclusive features between two clustered groups.

Abstract Background Ov16 serology is considered a reference method for Onchocerca volvulus epidemiological mapping. Given the suboptimal sensitivity of this test and the fact that seroconversion takes more than a year after infection, additional serological tests might be needed to guide onchocerciasis elimination programmes. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from Onchocerca volvulus non-endemic areas required further investigation. Methods We evaluated both peptide markers in an O. volvulus hypo-endemic setting in Jimma Town, Ethiopia using peptide ELISA. For all individuals (n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoni was known. Results We found that 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals were positive for OvMP-1 and OvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. No difference in seroprevalence for all three markers could be observed between uninfected individuals and individuals infected with different soil-transmitted helminths or S. mansoni. Moreover, the intensity of the response to OvMP-1, OvMP-23 or Ov16 was not significantly stronger in individuals carrying patent STH or S. mansoni infections, nor was there any correlation between the intensities of the responses to the three different antigens. Conclusions This study demonstrates that a patent infection with either soil-transmitted helminths or S. mansoni does not lead to increased antibody recognition of both OvMP-1 and OvMP23.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. We sought to allow description of the complexity of IgE, IgG 4 , and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG 4 , and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. Extensive induction of linear peptide-specific Phl p 1– and Bet v 1–specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Introduction The ability to predict epitopes plays an enormous role in vaccine development in terms of our ability to zero in on where to do a more thorough in-vivo analysis of the protein in question. Though for the past decade there have been numerous advancements and improvements in epitope prediction, on average the best benchmark prediction accuracies are still only around 60%. New machine learning algorithms have arisen within the domain of deep learning, text mining, and convolutional networks. This paper presents a novel analytically trained and string kernel using deep neural network, which is tailored for continuous epitope prediction, called: Deep Ridge Regressed Epitope Predictor (DRREP). Results DRREP was tested on long protein sequences from the following datasets: SARS, Pellequer, HIV, AntiJen, and SEQ194. DRREP was compared to numerous state of the art epitope predictors, including the most recently published predictors called LBtope and DMNLBE. Using area under ROC curve (AUC), DRREP achieved a performance improvement over the best performing predictors on SARS (13.7%), HIV (8.9%), Pellequer (1.5%), and SEQ194 (3.1%), with its performance being matched only on the AntiJen dataset, by the LBtope predictor, where both DRREP and LBtope achieved an AUC of 0.702. Conclusion DRREP is an analytically trained deep neural network, thus capable of learning in a single step through regression. By combining the features of deep learning, string kernels, and convolutional networks, the system is able to perform residue-by-residue prediction of continues epitopes with higher accuracy than the current state of the art predictors.

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery. To better understand the underlying mechanisms of antibody-antigen interaction, here, we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cow milk is a major allergenic food. The potential prevention and treatment effects of lactic acid bacteria (LAB)-fermented dairy products on allergic symptoms have garnered considerable attention. Cow milk allergy (CMA) is mainly attributed to extracellular and/or cell envelope proteolytic enzymes with hydrolysis specificity. Numerous studies have demonstrated that LAB prevents the risk of allergies by modulating the development and regulation of the host immune system. Specifically, LAB and its effectors can enhance intestinal barrier function and affect immune cells by interfering with humoral and cellular immunity. Fermentation hydrolysis of allergenic epitopes is considered the main mechanism of reducing CMA. This article reviews the linear epitopes of allergens in cow milk and the effect of LAB on these allergens and provides insight into the means of predicting allergenic epitopes by conventional laboratory analysis methods combined with molecular simulation. Although LAB can reduce CMA in several ways, the mechanism of action remains partially clarified. Therefore, this review additionally attempts to summarize the main mechanism of LAB fermentation to provide guidance for establishing an effective preventive and treatment method for CMA and serve as a reference for the screening, research, and application of LAB-based intervention., (© 2023 Institute of Food Technologists®.)

An IgE epitope is a part of an allergen that is capable of binding to IgE antibodies and eliciting an immune response. Identifying and characterizing human-allergy-relevant epitopes are important for diagnosis and prognosis of food allergy and development of immunotherapy treatments. This chapter describes the protocol for manual synthesis of overlapping peptides on a cellulose membrane and subsequent dot blotting of the peptides with allergic patients' IgE to map the linear IgE-binding epitopes in food allergens., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fish allergy is a life-long food allergy whose prevalence is affected by many demographic factors. Currently, there is no cure for fish allergy, which can only be managed by strict avoidance of fish in the diet. According to the WHO/IUIS Allergen Nomenclature Sub-Committee, 12 fish proteins are recognized as allergens. Different processing (thermal and non-thermal) techniques are applied to fish and fishery products to reduce microorganisms, extend shelf life, and alter organoleptic/nutritional properties. In this concise review, the development of a consistent terminology for studying food protein immunogenicity, antigenicity, and allergenicity is proposed. It also summarizes that food processing may lead to a decrease, no change, or even increase in fish antigenicity and allergenicity due to the change of protein solubility, protein denaturation, and the modification of linear or conformational epitopes. Recent studies investigated the effect of processing on fish antigenicity/allergenicity and were mainly conducted on commonly consumed fish species and major fish allergens using in vitro methods. Future research areas such as novel fish species/allergens and ex vivo/in vivo evaluation methods would convey a comprehensive view of the relationship between processing and fish allergy.

Linear B-cell epitope prediction research has received a steadily growing interest ever since the first method was developed in 1981. B-cell epitope identification with the help of an accurate prediction method can lead to an overall faster and cheaper vaccine design process, a crucial necessity in the COVID-19 era. Consequently, several B-cell epitope prediction methods have been developed over the past few decades, but without significant success. In this study, we review the current performance and methodology of some of the most widely used linear B-cell epitope predictors which are available via a command-line interface, namely, BcePred, BepiPred, ABCpred, COBEpro, SVMTriP, LBtope, and LBEEP. Additionally, we attempted to remedy performance issues of the individual methods by developing a consensus classifier, which combines the separate predictions of these methods into a single output, accelerating the epitope-based vaccine design. While the method comparison was performed with some necessary caveats and individual methods might perform much better for specialized datasets, we hope that this update in performance can aid researchers towards the choice of a predictor, for the development of biomedical applications such as designed vaccines, diagnostic kits, immunotherapeutics, immunodiagnostic tests, antibody production, and disease diagnosis and therapy.

Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA's and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox. [ABSTRACT FROM AUTHOR]

Type I collagen was described as a major allergen in fish. The purpose of this study was to screen and identify the linear IgE epitopes of type I collagen α1 and α2 subunits in rainbow trout. Five bioinformatics tools were used to predict the potential epitopes and the resultant epitopes were confirmed by LAD2 cells degranulation assay with sera from fish allergic patients. As the result, 10 peptides of α1 and α2 subunits were predicted, respectively, and these peptides were assembled by solid-phase synthesis. 14 epitopes were identified by LAD2 cells degranulation assay, among which, peptide 2, 5-7 were identified as linear epitope of α1 and peptide 11-20 were identified as linear epitope of α2. Moreover, for α1 and α2 subunits, the similarity of sequences was greater than 79%, suggesting the cross-reactivity of fish collagen. The findings of this study provided a strong support for further research of reduction of the collagen allergenicity. [ABSTRACT FROM AUTHOR]

Background: The Iridoviridae family is categorized into five genera and clustered into two subfamilies: Alphairidovirinae includes Lymphocystivirus, Ranavirus (GIV), and Megalocystivirus (TGIV), which infect vertebrate hosts and Betairidovirinae includes Iridovirus and Chloriridovirus, which infect invertebrate hosts. Clustered Iridoviridae subfamilies possess host-specific characteristics, which can be considered as exclusive features for in-silico prediction of effective epitopes for vaccine development. A voting mechanism-based linear epitope (LE) prediction system was applied to identify and endorse LE candidates with a minimum length requirement for each clustered subfamily Results: The experimental results showed that four conserved epitopes among the Iridovirideae family, one exclusive epitope for invertebrate subfamily and two exclusive epitopes for vertebrate family were predicted. These predicted LE candidates were further validated by ELISA assays for evaluating the strength of antigenicity and cross antigenicity. The conserved LEs for Iridoviridae family reflected high antigenicity responses for the two subfamilies, while exclusive LEs reflected high antigenicity responses only for the host-specific subfamily Conclusions: Host-specific characteristics are important features and constraints for effective epitope prediction. Our proposed voting mechanism based system provides a novel approach for in silico LE prediction prior to vaccine development, and it is especially powerful for analyzing antigen sequences with exclusive features between two clustered groups. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Ov16 serology is considered a reference method for Onchocerca volvulus epidemiological mapping. Given the suboptimal sensitivity of this test and the fact that seroconversion takes more than a year after infection, additional serological tests might be needed to guide onchocerciasis elimination programmes. Recently, two linear epitopes encoded in OvMP-1 and OvMP-23 peptides were introduced as serological markers, but the observed antibody cross-reactivity in samples originating from Onchocerca volvulus non-endemic areas required further investigation. Methods: We evaluated both peptide markers in an O. volvulus hypo-endemic setting in Jimma Town, Ethiopia using peptide ELISA. For all individuals (n = 303), the infection status with soil-transmitted helminths and Schistosoma mansoni was known. Results: We found that 11 (3.6%) individuals were positive for anti-Ov16 IgG4 antibodies, while 34 (11.2%) and 15 (5.0%) individuals were positive for OvMP-1 and OvMP-23, respectively. Out of the 34 OvMP-1 positive samples, 33 were negative on the Ov16 IgG4 ELISA. Similarly, out of the 15 OvMP-23 positive samples, 14 scored negative on this reference method. No difference in seroprevalence for all three markers could be observed between uninfected individuals and individuals infected with different soil-transmitted helminths or S. mansoni. Moreover, the intensity of the response to OvMP-1, OvMP-23 or Ov16 was not significantly stronger in individuals carrying patent STH or S. mansoni infections, nor was there any correlation between the intensities of the responses to the three different antigens. Conclusions: This study demonstrates that a patent infection with either soil-transmitted helminths or S. mansoni does not lead to increased antibody recognition of both OvMP-1 and OvMP23. [ABSTRACT FROM AUTHOR]

Highlights • A novel linear B cell epitope was identified at the C terminus of the helix A in HA2 of H7N9. • Sera against such epitope derived from H9, H7 and H3, not from H5 and H1, demonstrated cross-reaction between Group 1 and Group 2. • Engineering such epitope could provide novel targets for peptide based broad-spectrum influenza vaccine candidate. Abstract In this research, four monoclonal antibodies (mAbs) were first generated as an immunogen by using the GST fusion protein that carries the fusion peptide and helix A derived from H7N9 influenza A virus (IAV). These mAbs could react with HA of H7N9, H3N2, and H9N2 with neutralizing activity. A novel linear epitope recognized by these mAbs was identified by peptide-based ELISA, and this epitope was located in TAADYKSTQSAIDQITGKLN at the C terminus of the helix A of H7N9. 3 A11, which is one of the four mAbs, could efficiently recognize the corresponding epitopes derived from H9, H7, H5, H3, and H1. Analysis of sera against the corresponding epitope from different HAs revealed that the C terminus of helix A in H9, H7, and H3 possessed dominant B cell epitopes that cross both Group 1 and Group 2 IAV, whereas the C terminus of helix A in H5 possessed only dominant B cell epitopes that cross subtypes in Group 1 virus. All these results demonstrated that the linear epitope identified in the helix A of H7N9 could be a novel target for developing broad-spectrum influenza diagnostics or vaccine candidates. [ABSTRACT FROM AUTHOR]

The main immunogenic protein of the porcine epidemic diarrhea virus (PEDV) is the spike protein (S protein), which plays an important role in receptor binding, membrane fusion, and viral invasion of the host. In this paper, the linear epitope of the 3F10 non-neutralizing monoclonal antibody that was previously prepared in our laboratory was identified. The expression of truncated forms of the S protein for 3F10 reaction studies proved that the epitope was located between amino acids (aa) 674 and 791. To further locate the core aa of the 3F10 epitope, 12 random peptide libraries were used, and the result showed that the key aa located at aa 685-688 of the S protein (LLAF) were recognized by 3F10. Homology analysis of the regions corresponding to 20 typical strains of different PEDV subtypes showed that the epitope is highly conserved. Identifying the epitope recognized by an antibody helps to improve our understanding of the structure and function of the antigen. [ABSTRACT FROM AUTHOR]

Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition. [ABSTRACT FROM AUTHOR]