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Infection by the novel duck reovirus (NDRV) in ducklings causes high mortality, which leads to substantial economic losses in the duck industry in China. To date, no commercial vaccine is available for this disease. In this study, linear B cell epitopes of the σB protein of the NDRV were predicted and recombinant multiple linear B cell epitopes (MLBEs) were constructed through linkers. The recombinant MLBEs were then expressed and purified. One-day-old Muscovy ducklings were immunized with different doses of MLBEs and challenged with 5 × 104 ELD50 of the virulent CHY strain of NDRV 14 days after immunization. The ducklings vaccinated with 20 and 40 µg of MLBE performed no clinical signs or gross or histopathological lesions, indicating 100% protection against infection. The viral load in the liver and spleens of these birds was significantly lower than that in the control group. Additionally, these ducklings exhibited positive seroconversion at 7 days after vaccination on enzyme-linked immunosorbent assay. These results indicate that MLBE of σB could be used as a candidate for developing vaccines against NDRV infection.

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The viral proteins VP1-1, VP2, VP4, VP7 and NS3, of African horse sickness virus serotype 4 (AHSV4), have previously been identified to contain CD8+ T cell epitopes. In this study, overlapping peptides spanning the entire sequences of these AHSV4 proteins were synthesized and used to map epitopes. Peripheral blood mononuclear cells (PBMC) isolated from five horses immunized with an attenuated AHSV4 were stimulated in vitro with the synthesized peptides. Various memory immune assays were used to identify the individual peptides that contain CD8+ T cell epitopes, CD4+ T cell epitopes and linear B cell epitopes. The newly discovered individual peptides of AHSV4 proteins VP1-1, VP4, VP7 and/or NS3 that contain CD8+ T cell, CD4+ T cell or linear B cell epitopes could contribute to the design and development of new generation AHS peptide-based vaccines and therapeutics.

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Full-length nucleotide sequences of avian influenza A virus neuraminidase coding region (20,631 sequences) were analyzed and compared with those isolated from viruses infecting human and swine (63,750 sequences). If in fourfold degenerate sites there is asymmetric A-bias that may be more or less asymmetric depending on the type of neuraminidase and the host, than in twofold degenerate sites from third codon positions there is a strong asymmetric U-bias in coding regions of N4, N5, and N8 isolated from viruses infecting birds, as well as in those of N1 and N2 isolated from viruses infecting human, swine, and birds, while in coding regions of N9 isolated from birds, there is surprisingly strong C-bias, and in sequences of N3, N6, and N7 the usage of C is quite close to the level of U. Revealed stabilization of both U and C in twofold degenerate sites is the evidence of frequent changes in mutational pressure direction. Asymmetric mutational pressure was one of the sources of amino acid replacements that resulted in an equal percentage of sites with appeared and disappeared linear B-cell epitopes in N1, N2, N4, and N5 (33.62-35.33% vs. 32.41-36.45%, respectively), and controlled by the immune pressure it resulted in a stronger tendency to disappear for B-cell epitopes of N3, N6, N7, N8, and N9 of avian viruses (8.74-28.77% vs. 28.96-38.89%). The lack of correlation between nucleotide usages in fourfold and twofold degenerate sites for three nucleotides, except U, is a strong evidence of mutational pressure theory.

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Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear B-cell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1α, IL-1ß, IL-6, IL-8, and TNF-α. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1ß and IL-6, between genotype-specific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

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The accurate prediction of B cell epitopes is crucial for the design and development of vaccines, especially of those preventive for emerging pathogenic diseases. Preventive vaccines are mainly based on the induction of highly specific neutralizing antibodies. This chapter deals with some prediction methods, which are currently available as user-friendly online servers, to predict B cell epitopes in proteins. A final assessment to validate these predictions is done by recurring to the Immune Epitope Database (IEDB).

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Linear B-cell epitopes have a prominent role in the development of peptide-based vaccines and disease diagnosis. High variability in the length of these epitopes is a major reason for low accuracy in their prediction. Most of the B-cell epitope prediction methods considered fixed length of epitope sequences and achieved good accuracy. Though a number of tools are available for the prediction of flexible length linear B-cell epitopes with reasonable accuracy, further improvement in the prediction performance is still expected. Thus, here we made an attempt to analyze the performance of machine learning approaches (MLA) with 18 different amino acid encoding schemes in the prediction of flexible length linear B-cell epitopes. We considered B-cell epitope sequences of variable lengths (11-56 amino acids) from well-established public resources. The performances of machine learning algorithms with the encoded epitope sequence datasets were evaluated. Besides, the feasible combinations of encoding schemes were also explored and analyzed. The results revealed that amino-acid composition (AC) and distribution component of composition-transition-distribution encoding schemes are suitable for heterogeneous epitope data, whereas amino-acid-anchoring-pair-composition (APC), dipeptide-composition and amino-acids-pair-propensity-scale (APP) are more appropriate for homogeneous data. Further, two combinations of peptide encoding schemes, i.e. APC + AC and APC + APP with random forest classifier were identified to have improved performance over the state-of-the-art tools for flexible length linear B-cell epitope prediction. The study also revealed better performance of random forest over other considered MLAs in the prediction of flexible length linear B-cell epitopes.

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Feline calicivirus (FCV) is a common and important pathogen in cats, typically resulting in upper respiratory tract disease or ulcerative oral lesions. Although there are large number of researches on FCV and vaccines against FCV have been widely used for years, the explanation for vaccination failure and further studies on the prevalence of FCV are still necessary in China. In this study, 86 nasopharyngeal swabs from pet cats with upper respiratory symptoms from several Nanjing animal hospitals were collected in 2020. Among them, 36 (41.86%) were positive for FCV. In addition, 13 FCV capsid genes were sequenced. The comparative analysis of linear B-cell epitopes of VP1 gene indicated that there were many amino acid variations existed among FCV vaccine strains and these strains currently circulating in Nanjing, which may relate to the failure of vaccination and maybe aid for future vaccine design. Besides, phylogenetic analysis of capsid gene revealed two genotypes. Except for the F86 strain, most of the strains were clustered with FCV I genotype, which indicated that FCV I genotype was the most prevalent genotype currently circulating in Nanjing. In conclusion, this study provided useful information as to the evolution and genetic variants of FCV in Nanjing, which is urgent for the future instructions of effective disease prevention and control strategies.

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Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%. In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, 423FTCSQIS429 and 546SPLEGGGWL554,were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope 423FTCSQIS429 while residues 548L, 550G, 553W, 554L for epitope 546SPLEGGGWL554. These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods.

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The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

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COVID-19 caused by SARS-CoV-2 is sweeping the world and posing serious health problems. Rapid and accurate detection along with timely isolation is the key to control the epidemic. Nucleic acid test and antibody-detection have been applied in the diagnosis of COVID-19, while both have their limitations. Comparatively, direct detection of viral antigens in clinical specimens is highly valuable for the early diagnosis of SARS-CoV-2. The nucleocapsid (N) protein is one of the predominantly expressed proteins with high immunogenicity during the early stages of infection. Here, we applied multiple bioinformatics servers to forecast the potential immunodominant regions derived from the N protein of SARS-CoV-2. Since the high homology of N protein between SARS-CoV-2 and SARS-CoV, we attempted to leverage existing SARS-CoV immunological studies to develop SARS-CoV-2 diagnostic antibodies. Finally, N229-269, N349-399, and N405-419 were predicted to be the potential immunodominant regions, which contain both predicted linear B-cell epitopes and murine MHC class II binding epitopes. These three regions exhibited good surface accessibility and hydrophilicity. All were forecasted to be non-allergen and non-toxic. The final construct was built based on the bioinformatics analysis, which could help to develop an antigen-capture system for the early diagnosis of SARS-CoV-2.

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Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, and has caused huge losses throughout the world. The structural viral protein VP2, which is able to self-assemble into empty capsids, known as virus-like particles (VLPs), is crucial to induce PPV-specific neutralizing antibodies and protective immunity. In this study, twelve monoclonal antibodies (mAbs) against PPV were generated. The mAbs were characterized by indirect enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and virus neutralization (VN) assay. Two mAbs were defined to be able to neutralize the standard PPV 7909 strain. Subsequently, peptide scanning was applied to identify linear epitopes. The peptide, 89ESGVAGQMV97 was defined as a precise linear epitope. Results from structural analysis showed that the epitope was exposed on the virion surface. Multiple sequence alignment analysis indicated that peptide 89ESGVAGQMV97 was not completely conserved, with a higher amino acid mutation rate at 91G, 92V and 93A position. Alanine-scanning mutagenesis further revealed that residues 89E, 90S, 91G, 92V and 94G were the core sites involved in antibody recognition. These findings may facilitate further understanding the function of the VP2 protein and development of diagnostic tools.

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Neutralizing, serotype-specific, and hemagglutination-inhibiting antibodies against infectious bronchitis virus (IBV) are induced by epitopes in the S1 protein. Most changes in the virus genome due to mutation and recombination during serial passaging in embryonated chicken eggs occur in the S1 gene. In the current study, we tried to predict the potential linear B-cell epitopes of the S1 subunit of two Iranian 793/B isolates and then we analyzed their changes at passage level 90 due to mutations at this passage level. To predict linear B-cell epitopes of the S1 protein belonging to two Iranian 793/B isolates, we used two online epitope prediction programs called BepiPred and ABCpred. Some of the most important features of proteins including antigenicity, physicochemical properties, and secondary structure composition were analyzed. The predicted epitopes were studied between wild viruses and their passage level 90 viruses. We identified 15 potential linear B-cell epitopes among which six epitopes had the highest scores of physicochemical properties and antigenicity. Due to amino acid substitutions, seven predicted epitopes had different amino acid sequences at passage level 90. Among eight epitopes with no amino acid substitution at passage level 90, three epitopes had the highest scores. These three conserved epitopes including NH2-NQLGSCPLTGMI-COOH, NH2-GNFSDGFYPFTNSSLVKD-COOH, and NH2-GPIQGGC-COOH might be strategic and potential candidates for use in designing epitope-based vaccine researches. In conclusion, based on scores of physicochemical properties and antigenicity, it seemed that the sequence of most epitopes in wild viruses might be more antigenic and immunogenic compared to their sequence in viruses of passage 90.

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Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed "clean linear B cell epitopes," and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.

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Trypanosoma vivax is a parasite widespread across Africa and South America. Immunological methods using recombinant antigens have been developed aiming at specific and sensitive detection of infections caused by T. vivax. Here, we sequenced for the first time the transcriptome of a virulent T. vivax strain (Lins), isolated from an outbreak of severe disease in South America (Brazil) and performed a computational integrated analysis of genome, transcriptome and in silico predictions to identify and characterize putative linear B-cell epitopes from African and South American T. vivax. A total of 2278, 3936 and 4062 linear B-cell epitopes were respectively characterized for the transcriptomes of T. vivax LIEM-176 (Venezuela), T. vivax IL1392 (Nigeria) and T. vivax Lins (Brazil) and 4684 for the genome of T. vivax Y486 (Nigeria). The results presented are a valuable theoretical source that may pave the way for highly sensitive and specific diagnostic tools.

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Neglected tropical diseases caused by helminth infections currently affect millions of people worldwide. Among them, there are three tapeworm species of outstanding importance: Echinococcus granulosus, E. multilocularis, and Taenia solium, which are responsible for cystic echinococcosis, alveolar echinococcosis, and cysticercosis, respectively. Despite several attempts, there is still a need for an effective and low-cost serological diagnostic test that can be used in endemic countries. In the present work, we described an innovative bioinformatic workflow for a rational prediction of putative peptide candidates for one-step serological diagnosis of any of these infections. First, we predicted the theoretical secretome shared by the three tapeworms starting from their full reported proteomes. Then, through immunoinformatics, we identified proteins within the shared secretome displaying high antigenicity scores and bearing T cell epitopes able to bind most human MHC-II alleles. Secondly, in such proteins, we identified linear B cell epitopes without post-translational modifications, and mapped them on 3D modelled structures to visualize their antibody accessibilities. As a result, we finally suggested two antigenic peptides shared between the secretomes of the three parasite species, which could be further tested for their immunodiagnostic potential.

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BACKGROUND: Accurate identification of linear B-cell epitopes plays an important role in peptide vaccine designs, immunodiagnosis, and antibody productions. Although several prediction methods have been reported, unsatisfied accuracy has limited the broad usages in linear B-cell epitope prediction. Therefore, developing a reliable model with significant improvement on prediction accuracy is highly desirable. RESULTS: In this study, we developed a novel model for prediction of linear B-cell epitopes, APCpred, which was derived from the combination of amino acid anchoring pair composition (APC) and Support Vector Machine (SVM) methods. Systematic comparisons with the existing prediction models demonstrated that APCpred method significantly improved the prediction accuracy both in fivefold cross-validation of training datasets and in independent blind datasets. In the fivefold cross-validation test with Chen872 dataset at window size of 20, APCpred achieved AUC of 0.809 and accuracy of 72.94%, which was much more accurate than the existing models, e.g., Bayesb, Chen's AAP methods and the enhanced combination method of AAP with five AP scales. For the fivefold cross-validation test with ABC16 dataset, APCpred achieved an improved AUC of 0.794 and ACC of 73.00% at window size of 16, and attained an AUC of 0.748 and ACC of 67.96% on Blind387 dataset after being trained with ABC16 dataset. Trained with Lbtope_Confirm dataset, APCpred achieved an increased Acc of 55.09% on FBC934 dataset. Within sequence window sizes from 12 to 20, APCpred final model on homology-reduced dataset achieved an optimal AUC of 0.748 and ACC of 68.43% in fivefold cross-validation at the window size of 20. CONCLUSION: APCpred model demonstrated a significant improvement in predicting linear B-cell epitopes using the features of amino acid anchoring pair composition (APC). Based on our study, a webserver has been developed for on-line prediction of linear B-cell epitopes, which is a free access at: http:/ccb.bmi.ac.cn/APCpred/.